Since the domains also have well- known three-dimensional ..... transformation in that cell-free extract rather than intact cells are used for the biosynthesis of the ...
Protein engineering to explore and improve affinity ligands Martin Linhult
Royal Institute of Technology Department of Biotechnology Stockholm 2003
ISBN 91-7283-596-6 Stockholm 2003 © Martin Linhult Royal Institute of Technology Albanova University Center Department of Biotechnology SE-106 91 Stockholm Sweden Printed at Författares Bokmaskin S:t Eriksgatan 10 Box 12071 SE-102 22 Stockholm
Linhult M.2003. Protein engineering to explore and improve affinity ligands. Department of Biotechnology, Albanova University Center, Royal Institute of Technology, Stockholm, Sweden.
Abstract In order to produce predictable and robust systems for protein purification and detection, well characterized, small, folded domains descending from bacterial receptors have been used. These bacterial receptors, staphylococcal protein A (SPA) and streptococcal protein G (SPG), possess high affinity to IgG and / or HSA. They are composed of repetitive units in which each one binds the ligand independently. The domains fold independently and are very stable. Since the domains also have wellknown three-dimensional structures and do not contain cysteine residues, they are very suitable as frameworks for further protein engineering. Streptococcal protein G (SPG) is a multidomain protein present on the cell surface of Streptococcus. X-ray crystallography has been used to determine the binding site of the Ig-binding domain. In this thesis the region responsible for the HSA affinity of ABD3 has been determined by directed mutagenesis followed by functional and structural analysis. The analysis shows that the HSAbinding involves residues mainly in the second a-helix. Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is the preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. Here, a protein engineering strategy has been used to improve the tolerance to alkaline conditions of different domains from protein G, ABD3 and C2. Amino acids known to be susceptible towards high pH were substituted for less alkali susceptible residues. The new, engineered variants of C2 and ABD shown higher stability towards alkaline pH. Also, very important for the potential use as affinity ligands, these mutated variants retained the secondary structure and the affinity to HSA and IgG, respectively. Moreover, dimerization was performed to investigate whether a higher binding capacity could be obtained by multivalency. For ABD, binding studies showed that divalent ligands coupled using non-directed chemistry demonstrated an increased molar binding capacity compared to monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using a directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a well known and thoroughly characterized fusion partner widely used in affinity chromatography systems. This domain is considered to be relatively tolerant towards alkaline conditions. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning in place (CIP) procedures. For this purpose a different protein engineering strategy was employed. Small changes in stability due to the mutations would be difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, a by-pass mutagenesis strategy was utilized, where a mutated structurally destabilized variant, Z(F30A) was used as a surrogate framework. All eight asparagines in the domain were exchanged one-by-one. The residues were all shown to have different impact on the alkaline tolerance of the domain. By exchanging asparagine 23 for a threonine we were able to remarkably increase the stability of the Z(F30A)-domain towards alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold we were able to detect an increased tolerance towards alkaline treatment compared to the native Z molecule. In all cases, the most sensitive asparagines were found to be located in the loops region. In summary, the work presented in this thesis shows the usefulness of protein engineering strategies, both to explore the importance of different amino acids regarding stability and functionality and to improve the characteristics of a protein.
Keywords: binding, affinity, human serum albumin (HSA), albumin-binding domain (ABD), affinity chromatography, deamidation, protein A, stabilization, Z-domain, capacity, protein G, cleaning-in-place (CIP), protein engineering, C2 receptor. © Martin Linhult
An intellectual is a man who says a simple thing in a difficult way; an artist is a man who says a difficult thing in a simple way. Charles Bukowski, “Notes of a Dirty Old Man”
List of publications This thesis is based on the following publications, referred to in the text by their respective roman numerals: I.
Linhult, M., Binz, K., Uhlén, M. and Hober, S. (2002) Mutational analysis of the interaction between Albumin-Binding Domain (ABD) from Streptococcal protein G and Human Serum Albumin (HSA). Protein Sci. 11: 206-213.
II.
Gülich, S., Linhult, M., Nygren, P-Å., Uhlén, M. and Hober, S. (2000) Stabilization of an affinity protein ligand towards Cleaning In Place (CIP) conditions. J Biotechnol. 80:169-78.
III.
Linhult, M., Gülich S., Gräslund, T., Nygren, P-Å. and Hober, S. (2003) Evaluation of different linker regions for multimerization and the coupling chemistry for immobilization of a proteinaceous affinity ligand. Protein Engineering In press.
IV.
Gülich, S,. Linhult, M., Ståhl, S. and Hober. S. (2002) Engineering Streptococcal protein G for increased alkaline stability. Protein Engineering 15(10):835-42.
V.
Linhult, M., Gülich, S., Gräslund, T., Simon, A., Karlsson, M., Sjöberg, A., Nord, K. and Hober, S. (2003) Improving the tolerance of protein A to repeated alkaline exposures using a by-pass mutagenesis approach. Proteins: Struct., Funct. and Genet. In press.
Table of contents Introduction
10
Protein structure
11
Protein engineering
13
Site directed mutagenesis
13
Combinatorial approaches
15
Protein stability
17
Rational design
20
Tolerance to alkaline conditions
22
Protein-protein interactions
24
Kinetic studies
24
Measuring protein-protein interactions
26
Bacterial surface receptors
27
Staphylococcal protein A
28
Streptococcal protein G
30
HSA binding region
30
IgG binding region
32
Affinity chromatography purification
33
Present investigation
36
HSA binding domain
36
Mutational analysis of an ABD
36
Stabilization of ABD towards alkaline conditions
40
IgG binding domains
49
Stabilization of C2 towards alkaline conditions
49
Stabilization of Z towards alkaline conditions
53
Concluding remarks
58
Acknowledgements
59
Abbreviations
60
References
61
Introduction Fifty years have now passed since the structure of the DNA double helix was discovered (Watson and Crick 1953). In this time, great efforts have been made in the field of life sciences, leading to advance across a range of important and emerging areas. A continued challenge for scientists in the coming years will be to further deepen our understanding of biology at the molecular level, including all classes of biomolecules involved in life processes. An increased knowledge about protein function in particular has become especially important in the so-called post genomic era, where many unknown gene products are currently being characterized for localization, expression levels, interactions and ultimately function. Somewhat simplified, proteins are linear biomolecules of different lengths which are synthesized from 20 different amino acid building blocks by the cellular machinery according to a blueprint stored as genetic information. Following synthesis, the linear molecule undergoes complex folding processes forming various secondary structure elements such as a-helices, b-sheets and different turn/loops. Subsequent arrangement of these structure elements results in the final three-dimensional structure of a protein. Despite the limited number of building blocks, proteins can be very diverse with respect to for example their functions, and the environments in which they act. Fundamental questions to answer in this respect are for example how proteins have evolved to interact with other proteins and to be function in radically different environments. Protein engineering is one approach that can be used to shed some light onto these questions, and increase the utility of the proteins in different biotechnological and industrial applications. Protein engineering techniques have proven fruitful for resolving questions in the field of biochemistry since the late 1970s; pioneered by the work done on site-directed mutagenesis on Tyrosyl-transfer RNA synthetase (TyrS) by Michael Smith and co-workers in Vancouver (Hutchison et al. 1978; Winter et al. 1982). In recent years, protein engineering has evolved from studies involving the substitution, deletion or insertion of amino acids one-by-one, to studies involving the manipulation of larger parts of proteins. This has become possible through the development of a range of new techniques, for instance the polymerase chain reaction (PCR) facilitating genetic work, gene shuffling for generation of diversity and different in vitro selection techniques, such as phage display. At its inception, protein engineering was used as a tool for understanding more about protein structure and 10
function. More recently, a number of different applications have come into existence, proving the success of protein engineering outside the lab. For instance, proteases and lipases have been engineered to work well in modern washing machines, and antibodies are now being engineered for clinical use in therapeutic applications (Holliger and Bohlen 1999; McCafferty and Glover 2000).
Protein structure The building blocks of proteins and peptides are the amino acids linked together with planar peptide bonds, described by Pauling as early as 1960 (Pauling 1960). The 20 natural amino acids differ in their respective properties, such as hydrophobicity, size, shape, and electrostatics. By altering the combinations and numbers of these amino acids, nature can build an immense number of more or less complex protein structures, of different stabilities and functions. The different properties of amino acids make them more or less suited to be present in different types of secondary structure elements (usually referred to as having different helix or b-sheet propensities). A number of studies have been performed to explore such different amino acid propensities in a-helices and b-sheets (Lyu et al. 1990; O'Neil and DeGrado 1990; Horovitz et al. 1992; Blaber et al. 1993; Munoz and Serrano 1994; Smith et al. 1994; Smith and Regan 1995; Myers et al. 1997). In addition, also loops and turns have been investigated in relation to amino acid propensities (Williams et al. 1987; Wojcik et al. 1999; Crasto and Feng 2001). These results can be beneficial in protein engineering, giving advice on which amino acids could be introduced in what structure elements without disturbing the overall structure. A popular strategy to investigate the importance of a particular amino acid position for a given trait is to replace the native amino acid with an alanine. The characteristics of this amino acid (relative small, pH-independent properties, high ahelix and b-sheet propensities) make it a good first choice in site-directed mutagenesis approaches (Fersht 1999). The a-helix consists of a right-handed coil wherein the n residue forms a hydrogen bond with the n+4 residue, resulting in 3.6 residues per turn. Hence, neither the first nor the last residues in the a-helix can make the intra-helical hydrogen bond between the backbone C=O groups of one turn and the NH groups of residues in the next (Fersht, 1999). This requires that for stabilization of the helix, hydrogen bonds must 11
be made with either other groups in the protein or the solvent. Capping boxes, termed N- and C-caps are often occurring in the N- and C-terminal to stabilize the ends of the helices. These residues form side chain hydrogen bonds to the backbone of the polypeptide chain inside the helix (Richardson and Richardson 1988; Doig 2002). The alignment of the dipoles in the polypeptide backbone causes a net dipole moment along the helix. The N-terminal is positively polarized while the C-terminal is negatively polarized Thus, there is a preference for negatively charged N-terminal amino acids, and consequently a preference for positively charged C-terminal amino acids (Hol 1985; Chakrabartty et al. 1993). Attempts to stabilize a-helices via favoring the electrostatics has been successful, for example, the introduction of negatively charged amino acid in the N-terminus of an a-helix of the T4 Lysozyme protein resulted in a stabilization as determined by (Nicholson et al. 1988). Polypeptide chains can also form complementary hydrogen bonds to a parallel polypeptide chain. These parallel chains can be aligned in either the same or opposite directions. This class of secondary structure elements is called the b-sheet. Because of the nature of b, sheets-always forming a hydrogen bond with another polypeptide chain, it is more difficult to predict b-sheet occurrence and amino acid propensities for b-sheet than for a-helices (Minor and Kim 1994b; a). Interestingly, high quantities of b-sheets occur in proteins prone to form arrays of insoluble fibrils, which are involved in for instance prion diseases such as Creutzfeldt-Jakob Disease (CJD) (Prusiner 1998). Loop and turn regions connect the a-helices and b-sheets. The definitions of turns and loops are not very distinct, with turns consisting of a few residues while loops are longer and have less well-defined structures. Polar and charged residues quite often occur on these surface exposed areas (Leszczynski and Rose 1986; Argos 1990). The structures described above constitute secondary structure elements of a protein. The tertiary structure of a protein is in turn defined by how these secondary structures of a single polypeptide chain are grouped together. A number of proteins consist of two or more polypeptide chains. The three dimensional arrangement of the different domains is in turn called the quaternary structure. Our understanding of the function/structure relationship of a protein is obviously facilitated if a three-dimensional structure of the protein is available. To date (sept 2003) 20 413 protein, peptide and virus structures have become available in the 12
Brookhaven Protein Databank; 17 653 which have been solved by X-ray crystallography and 2760 which have been solved by nuclear magnetic resonance (NMR). Another technique used to investigate protein structure is Circular Dichroism (CD) (Pace 1997a), a spectroscopy-based technique that measures the differential absorption of left and right circularly polarized light. The CD technique quickly and easily provides information about the secondary structure content of a protein sample (Schmid 1997; Kelly and Price 2000). CD spectroscopy is widely used as a tool in protein engineering, since it rapidly can assess the secondary structure of a range of protein constructs. By superimposing the spectra of different mutants, it is possible to verify structural differences. Another advantage with CD is that it is applicable in different experimental conditions, allowing for the collection of spectra in for example, different concentration of denaturants, different pHs and at different temperatures which can be used to analyze the stability of a protein (Kelly and Price 2000). The main limitation of CD is that it only provides relatively low-resolution structural information, but the conclusions can be enhanced through using secondary structure prediction algorithms (Ribas De Pouplana et al. 1991; Unneberg et al. 2001).
Protein engineering Site directed mutagenesis Site-directed mutagenesis has proven to be a valuable tool for increasing our understanding of different biomolecular principles, for instance enzyme mechanisms, folding pathways and biomolecular recognition. By changing a limited number of amino acids, and then investigating how these new amino acids influence the inherited function, the contribution of the various building blocks can be mapped. A high-resolution three-dimensional structure of the protein of interest (both the wild type and the variants) will obviously facilitate the evaluation of the collected data. However, even with the accumulated knowledge from numerous studies of different proteins, it is still very difficult, if not impossible to make accurate predictions about the effects, even from a single amino acid substitution in a protein. Therefore proteinengineering strategies could turn out to be a rather laborious. However, when both the three-dimensional structure is known, and the active part of the target protein is mapped, then the capacity for predicting the effect is greater. The first example of site
13
directed mutagenesis was presented by Michael Smith and co-workers in 1978. In this experiment, single-stranded viral DNA from an Escherichia coli (E. coli) specific bacteriophage was used as a template for mutagenesis. A mismatch oligonucleotide consisting of 12 bases was added and after hybridization, DNA polymerase I was used to make the DNA double stranded (ds). After ligation using T4-DNA ligase, the vector was transfected into E.coli cells resulting in the production of phages exhibiting the desired mutation (Hutchison et al. 1978). However, since both strands (wild type and mutation-containing strand) can serve as templates for replication, several clones must often be screened before a “pure” mutant is found. Later, Kunkel and coworker developed a more practical system (Kunkel et al. 1987), wherein the template vector first is propagated inside a uracil n-glycsoylate deficient (ung-) strain. This strain, to some extent incorporates uracil nucleotides instead of thymidines nucleotides into newly synthesized DNA. The heteroduplex DNA containing DNA with uracil in the template strand and newly synthesized DNA in the second strand is used to transfect an ung+ strain. The strain will efficiently degrade the template DNA strand, as it has incorporated uracil nucleotides. This can dramatically increase the mutation frequency (Kunkel et al. 1987; Kunkel et al. 1991). In the mid 1980s Kary B Mullis invented the PCR technique for enzymatic in vitro amplification of DNA. Variants of this technique have later been developed for site-specific mutagenesis purposes (Figure 1). In these methods PCR is performed with primers including the mutations to be inserted resulting in a PCR products containing the desired mutation.
14
A
a
c
b
d
B
C a
d
D
Figure 1. A PCR-based method for site-directed mutagenesis developed by Ho and coworkers (Ho et al. 1989), where (A) shows two separate PCR reactions which are performed with primers (a+b) and (c+d) respectively on the same template where c and b includes the desired mutation. B shows the two PCR products which both contain the inserted mutation. C shows the use of the previous produced PCR product as templates in a second PCR reaction using primers a and b resulting in a full-length product harboring the mutation (D).
Combinatorial approaches An alternative way to change the properties of a protein would be to create a library consisting of numerous member entities, and then carry out a selection or a screening procedure to find a member with desirable properties. Over the last few years, several techniques to create protein libraries have been developed providing links between genotype and phenotype, thereby facilitating the identification of the selected members by sequencing of the encoding gene. Phage display technology has to date been the most commonly used system for selection (Clackson and Wells 1994), which is described in the next paragraph. Recently a number of in vitro systems have been developed, for instance, ribosomal display (Hanes and Pluckthun 1997) and water-inoil emulsion selection (Tawfik and Griffiths 1998). One significant advantage with these systems is that they have overcome the library size-limiting step of
15
transformation in that cell-free extract rather than intact cells are used for the biosynthesis of the library members. The first phase when using protein-library methods are to create a gene library encoding for the different members. Several different methods have lately been reported in the literature for the generation of diversity in a library. One strategy is to employ synthetic oligonucleotides to code for a part of the gene. Here parts of the oligonucleotide are randomized to create diversity. Error-prone PCR is another widespread technique in which a thermostable DNA polymerase is forced to misincorporate nucleotides under certain conditions, such as the addition of Mn2+ (Miyazaki and Arnold 1999). In 1994 Stemmer described another strategy for creating diversity involving DNA shuffling. In this method, homologous genes are digested and reassembled using PCR to yield a pool of new genes (Stemmer 1994). Once the library containing the different gene constructs is established, it needs to be analyzed to find a member with the desired properties. This procedure can be performed using a screening or selection procedure. In the screening procedure, each member in the library is analyzed at the time. One elegant screening method which have been developed by Phillips and co-workers use the in vivo folding properties to screen for variants with an increased thermodynamic stability. The system is based on detection of protein folding in vivo and makes use of the distance-dependent fluorescence resonance energy transfer (FRET) from the green fluorescent protein (GFP) to the blue fluorescent protein (BFP). FRET only occurs when BFP and GFP are in close proximity. The system is based on the construction of a ternary fusion protein in which a protein of interest (X) is fused between GFP and BFP. If protein X is unfolded and degraded by cellular proteases FRET between the donor BFP and the acceptor GFP does not occur (Philipps et al. 2003). Working with selection, the optimal goal is to design a selection procedure in which only the variants with desired properties are isolated. Phage display is a powerful selection method applicable to both proteins and peptide libraries. The principle was first described in 1985 for the display of EcoRI endonuclease fragments on the surface of the E. coli filamentous phage M13 (Smith 1985). Phage display enables a physical link to be established between the protein of interest and the encoding genetic material. A foreign gene sequence is fused to the gene for one of the phage coat proteins, in most cases pIII or pVIII, making a hybrid fusion protein displayed on the surface of the phage. Thus when constructing a combinatorial library 16
of the protein or peptide of interest, each member exhibiting a unique property will be accessible on the surface of the phage enabling “fishing out” of the protein with the desired properties. Any method capable of separating clones with a desired trait from their background can be used as a selection method. In principle, most selection methods are based on affinity for a certain target molecule. After binding the phages to the target, non-specific binders are washed away. The phages that remain bound are then eluted and subsequently amplified. The amplified phages are again presented for the target, while non-specific binders are washed away. This routine is then repeated four to six times to find the desired proteins linked to the encoding DNA by phage particles (Clackson and Wells 1994).
Protein stability Over the last few years, stabilization of proteins has been of great interest to scientists for academic as well as economic reasons. When discussing protein stability one should bare in mind that different environment will affect the protein of interest differently. Proteins that have high thermal stability are not necessarily stable in high concentration of different unfolding agents, such as Urea, GdnHCl or alkaline solutions. Many attempts have been made to understand the structural and chemical reasons for the higher stability of enzymes isolated from extremozymes, in terms of their three-dimensional (3D) structure, than enzymes adapted to physiological environments. A structural comparison between mesophilic and extremophilic enzymes could illuminate ways for identifying mutations that could lead to stabilized variants. In some cases this has indeed been possible (Eijsink et al. 1995; Perl et al. 2000; Delbruck et al. 2001). Notable structural differences between mesophilic and thermophilic enzymes could for instance include increased compactness, shorter surface loops, smaller internal cavities and large ion-pair networks, as in the case for the enzyme citrate synthase (Danson and Hough 1998). However, using this knowledge in stabilizing proteins has proven to be more difficult than expected. The notable differences, which could be used as rules to stabilize mesophilic proteins, have been shown to not be generally applicable, and are therefore definitely not universal (Van den burg and Eijsink 2002). In an evolutionary context of adaptations to high temperatures, it can be kept in mind that the hyperthermophilic archaea represent the shortest phylogenetic lineage to 17
the root of the universal phylogenetic tree derived from small subunit (SSU) rRNA sequence data (Pace 1997b) (see Figure 2). Therefore stability, or at least thermostability, could be regarded as a starting point for evolution. Hence, by studying proteins from this source it might be possible to adapt numerous proteins for high temperature environment.
Figure 2. Universal phylogenetic tree based on SSU rRNA sequences. The scale bar corresponds to 0.1 changes per nucleotide. Reproduced, with permission from Pace (1997).
Thermostabilization of proteins would be advantageous for a number of reasons; for instance, an increased thermal stability would be beneficial for many industrial applications. A process running at a higher temperature would be accompanied by 18
higher reaction rates, higher solubility of reactants and a lower risk of microbial contaminations (van den Burg and Eijsink 2002). It is possible to increase the tolerance for a protein by adding extrinsic factor, such as glycine betaine. Glycine betaine has been found to stabilize a firefly luciferase used in bioluminometric assays at elevated temperature (Eriksson et al. 2003). Extrinsic factor seems also play a role in nature for different thermophilic bacteria. There is increasing evidence that hyperthermophilic archaea (archaebacteria) produce organic solutes such as 2-O-b-mannosylglycerate at high temperature (Hensel 1988; Martins 1997). Studies have also actually shown that these factors could have a direct effect on enzyme thermostability (Ramos 1997). In the field of protein stabilization through protein engineering, three different main strategies could be outlined, as random mutagenesis, consensus approaches and rational design. Recently, several groups have shown via different combinatorial techniques that it is possible to stabilize a protein by random mutagenesis. For instance, directed evolution, error-prone PCR, and gene shuffling are all techniques that have led to the creation of many new biocatalysts, including variants with significantly improved stability (Gershenson and Arnold 2000; Arnold 2001; Bornscheuer and Pohl 2001; Chen 2001). The common theme in these strategies is the creation of a library from which a suitable variant could be selected. The selection procedure must be high-throughput as well as sufficiently discriminative. Preferably, assay conditions are such that only stabilized variants are active, thereby enabling them to be detected. For example, selections for a protein that is essential for growth and survival of the host can be carried out in a thermophile (Gershenson and Arnold 2000). Another elegant selection strategy which can be mentioned here is one designed by Sieber with co-workers using a phage display system (Sieber et al. 1998). The general concept for the system is that an unfolded polypeptide chain, positioned C-terminally to one of the domains of protein III in a multidomain fusion protein displayed on the phage particle, would be cleaved through proteolysis. Hence, phage clones for which this occurs will loose the infectivity.
19
Rational design Rational design strategies have proven to be successful in making a protein more tolerant to different environments, such as increased temperature, extreme pHs and proteolytic enzymes. Traditionally, the most common strategy for increasing the structural stability for stabilizing a protein has been to change residues internally in the hydrophobic core (Van den Burg and Eijsink 2002). As the knowledge about protein structure and stability has increased, changing residues at the surface has turned out to be more successful strategy, since the hydrophobic core in most cases already is well packed. Difficulties in predicting the effects of one or more of the changed amino acids in the structurally important hydrophobic core may result in a less stable protein, or even a ruined scaffold. Furthermore, as the hydrophobic core is most often very well packed, the possibility to improve the stability by surface mutations, which instead influence the electrostatic interactions on the surface, is higher (Van den Burg and Eijsink 2002; Grimsley et al. 1999; Martin et al. 2001). In spite of the degree of unpredictability a number of rational approaches for structural stabilization have proven successful for a variety of proteins. These approaches include the improvement of the packing of the hydrophobic core, the stabilization of a-helix dipoles, the engineering of surface salt bridges and point mutations aimed at reducing the entropy of the unfolded state (Nicholson et al. 1988; Van den Burg et al. 1998; Grimsley et al. 1999; Wang et al. 1999; Olson et al. 2001). By comparing the amino acid content between two highly homologous domains, Serrano and co-researchers presented a rational design strategy for stabilizing a protein (Serrano et al. 1993). In this approach, each individual amino acid that differs between the two domains is mutated. Afterwards, each mutated protein is analyzed and the mutations that increase the stability can be combined to construct a multiple mutant. However, this strategy is quite tedious and has to be carried out carefully, as probably only a few key mutations will contribute to the increased stability (Van den Burg and Eijsink 2002). Eijsink and co-workers outlined another interesting alternative strategy in which a reduction of local unfolding could be of importance for increasing the thermal stability. For a neutral protease from Bacillus, it was found that stabilizing mutations were all clustered in a certain surface region of the protein (Van den Burg et al. 1998; Vriend et al. 1998). Further stabilization of the protein was found to be quite
20
straightforward as this region had been localized (Mansfeld et al. 1997; Van den Burg et al. 1998). The consensus approach, which is a semi-rational approach developed by Lehmann and co-researchers, can be regarded as an expansion of Serrano’s rational design of protein stabilization (Serrano et al. 1993; and previous section). This strategy is built around the idea that the amino acids contributing to the stability of the protein acid is retained during evolution and thereby a consensus residue should contribute more to the stability of a protein than a non-consensus amino acid. Therefore, while comparing the amino acid sequences of different homologous proteins, the most frequently occurring residue should be used (Lehmann et al. 2000; Lehmann and Wyss 2001; Lehmann et al. 2002). This strategy has turned out to be successful for obtaining a highly thermostable fungal phytases (Lehmann et al. 2000) and a more thermostable a-helical tetratricopeptide repeat (TPR) protein than its natural counter part (Main et al. 2003). Increasing the stability of proteins to proteases is also of interest in industrial applications of enzymes. If the proteolytic pattern is specific, it would be relatively easy to pinpoint the sensitive regions of the protein, and thus by rational design, alter the amino acid content and stabilize the protein. In some cases, while a higher temperature will unfold the proteins, the proteolytic stability correlates with the thermal stability due to the exposure of flexible amino acid sequences (McLendon and Radany 1978; Daniel et al. 1982; Parsell and Sauer 1989; Akasako et al. 1995). However, even folded proteins could be attacked by proteases if they contain regions that are accessible and flexible (Price 1990; Hubbard 1998). There are two different foci for altering amino acids to increase their tolerance to proteolysis; i.e decrease the conformational flexibility of the attacked structural region or changing the primary structure and introducing amino acids that are disadvantageous for the protease (Frenken et al. 1993; Markert et al. 2001).
21
Tolerance to alkaline conditions Changing the primary structure by introducing amino acids that could increase the tolerance of the protein towards harsh environment, such as temperature, pH or proteases, is a general concept for some of the work presented in this thesis. Asparagines are known to be susceptible to high pH, through covalent modifications such as deamidation (see path a in Figure 3) or backbone cleavage (see path b in Figure 3). Since NaOH is a common agent used to clean chromatographic media in large-scale processes (for a more detailed description, see the section Affinity chromatography) it is of importance for affinity ligands suitable for coupling to chromatographic matrices to be tolerant to high pH. Glutamine is also susceptible, though it is modified to a lesser extent than asparagine. These reactions are spontaneous and may also occur at physiological solvent conditions, often resulting in the loss of activity of the protein or peptide (Geiger and Clarke 1987). The extent of modification of the different residues is highly sequence and conformation dependent (Kossiakoff 1988; Lura and Schirch 1988; Kosky et al. 1999). The deamidation reaction involves the main chain peptide nitrogen succeeding the asparagine. The nitrogen functions as the nucleophile and attacks the side-chain carbonyl of asparagine, resulting in a succinimide intermediate. This succinimide intermediate may open at either of the two C-N bonds to form aspartic acid or isoaspartic acid, resulting in an addition of a negative charge. These two isomers may occur in their L or D forms (see path a in Figure 3). Cleavage of the peptide chain could also occur at the asparagine residues. In these reactions the mechanism proposed involves the side-chain amide nitrogen attacking the Ca, forming a C-terminal succinimide, as illustrated in path b in Figure 3. Both reactions are believed to occur simultaneously, although in general the deamidation reaction is faster (Tyler-Cross and Schirch 1991). An interesting hypothesis has been proposed by Robinson, in which through the reaction mechanisms presented above, glutaminyl and asparaginyl residues in peptides and proteins act as molecular timers of biological events such as protein turnover, development, and aging (Robinson et al. 1970; Robinson 1974; Robinson and Rudd 1974; Robinson and Lubke 1978; TylerCross and Schirch 1991).
22
O C NH2
H2C H2C
NH
RNH
C
RN+1
O
a
b
Asparagine
O
O
C
C
H2C
H2C NH
RN+1
NH2
H2C RNH
RNH
C O
Succinimide
O
O
C
C
O
NH
H2C
RN+1
H2C RNH
C O
Succinimide
H2C
NRN+1
H2C
H2C
NH C O
RN+1
Aspartate
O
RNH
C O
isoAspartate
Figure 3. Reaction mechanism for deamidation and backbone cleavage of asparagine residues in peptides and proteins via the formation of a succinimide intermediate; where in a the five-member succinimide intermediate is formed via a nucleophilic attack on the sidechain carbonyl carbon of asparagine by the backbone nitrogen of the ensuing amino acid, releasing NH3. The succinimide ring is labile to bases and readily opens, yielding either aspartic acid or isoaspartic acid. In b, peptide cleavage could also occur through a succinimide intermediate by attacking the side-chain amide of asparagine on the backbone peptide carbonyl of asparagine.
23
Protein-protein interactions Many biological processes are regulated through protein interactions involving noncovalent associations, for example signal transduction, cell regulation, immune response, and regulation of enzymatic activities. Apart from basic science aspects, a deeper understanding of the underlying principles of such biomolecular interactions would be helpful for the design of improved or novel affinity proteins. In addition, accurate prediction of binding surfaces, or hotspots from a three-dimensional structure (Ma et al. 2001; DeLano 2002) would be beneficial for the design of either agonist or antagagonist compounds for therapeutic applications. In general, hotspots residues in protein-protein interaction are defined as residues that upon mutation cause a large shift in binding affinity.
Kinetic studies A specific, rapid association of protein complexes is essential for a range of diverse processes. The rate of association of a protein complex is diffusion controlled, and in a random collision theory, the rate constant could be up to approximately 109 M-1 s-1 (Schreiber and Fersht 1996; Schreiber 2002). Most determined association rates are some thousands times lower, but association rates close to the diffusion limit have been measured (Fersht, 1999). In these cases, the speed of the process is functionally important, and introduction of additional electrostatic forces has been used to enhance the rate of association (Schreiber and Fersht 1996). The association of a pair of proteins can be described as a two-step reaction, A+B ‹=› [AB]* ‹=› AB, where A and B are the free proteins, [AB]* is the encounter complex, and AB is the final complex. The two proteins, A and B tumble randomly around until they reach an area designated the steering region. These collisions create a transition state that consequently produces the energetically favoured complex AB. The mechanisms behind such complex formation have been a matter of considerable research (Northrup and Erickson 1992; Janin 1997; Gabdoulline and Wade 1999; Selzer and Schreiber 2001). Many different strategies can be used to increase our understanding of the kinetics of protein-protein interactions, for instance, adding cosolvents, introducing mutations and changing the temperature or pH. The surroundings of the molecules are of great importance for the relative association rate; for example a simple relationship between 24
kon and viscosity with a slope of ~1 has been demonstrated (Raman et al. 1992; Stone and Hermans 1995). This means that the relative association rate (koon/k
v on)
in the
absence and presence of a viscous agent is linearly dependent on the relative viscosity (h/ho). In addition, ionic strength and pH are of obvious importance for the association of two biomolecules and different studies have shown a relationship between kon and ionic strength which follow the Debye-Hückel equation (Vijayakumar et al. 1998; Selzer and Schreiber 1999). Measuring the effect of mutation is a powerful tool that can be used to interpret protein-protein interactions. Extensive mutagenesis of the surface residues of different binding partners, such as TEM1-BLIP, barnase-barstar, interferon-receptor, growth hormone-receptor and interleukin-4-receptor, has demonstrated that mutations involving charged residues significantly affect the kon, whereas mutations of uncharged residues do not (Schreiber and Fersht 1995; Wang et al. 1997; Clackson et al. 1998; Albeck and Schreiber 1999; Piehler et al. 2000; Selzer et al. 2000). When introducing a substitution into a given protein, careful consideration has to be paid to the structural changes the new residue will generate. Hence, it is essential to carry out a structural comparison between the wild-type protein and the new mutated version. To ascertain whether the change in affinity upon mutation of two different residues is energetically independent on each other, employing a double mutant cycle is an effective strategy. Double mutant cycles were introduced in 1984 by Carter and coresearchers to study structural changes (Carter et al. 1984) changes. Later studies have shown that this strategy also is suitable for investigating protein-protein interactions (Schreiber and Fersht 1995; Frisch et al. 1997). By comparing the difference change in the free energy (DDG) upon complex formation between the single mutants (A and B) and HSA and the double mutant (C) and HSA (DDG=DGC-DGA-DGB), a synergistic effect introduced by the mutations can be detected. If the effects of the two single mutations are independent (which means DDG = 0), the residues do not effect each other; otherwise DDG ≠ 0, which means that the mutations are dependent on each other. One very interesting benefit arising from this knowledge is how the association rate is dependent on the electrostatic interactions. Using rational design, it has been shown possible to design faster association without affecting the dissociation (Selzer et al. 2000). The method is based on increasing the electrostatic attraction between the 25
proteins by incorporating charged residues in the vicinity of the binding interface. Improving the steering region, by changing the electrostatic interaction between the molecules, the association phase changes from random diffusion to directional movement towards the complex formation. This could be a more advantageous approach for enhancing binding affinity than affinity maturation by creating a library and going through a selection procedure. How important is the association and dissociation for the biological activity in the different events? A number of previous studies have indicated that the individual kinetic constants are indeed important; for example the human growth hormone receptor (hGH) requires an increase in dissociation of 30 fold compared to its wild type in order to bring about a linear dependence in EC50 for cell proliferation (Pearce et al. 1999). Similar results have also been observed for interleukin-4 and interferon (Piehler et al 2000; Wang et al 1997).
Measuring protein-protein interactions A number of techniques currently exist for investigating protein-protein interactions. Determination of the binding region through structural analysis of co-complexes, by for example X-ray crystallography and nuclear magnetic resonance (NMR), are two examples (Deisenhofer 1981; Wider and Wüthrich 1999). Measurements of the strengths of interactions between the two proteins could be done for example with isothermal titration calometry (ITC), surface plasmon resonance (SPR) and optical spectroscopy (Lakey and Raggett 1998). In combination with site-directed mutations, all of these could provide an excellent platform for investigating interactions. One technique for measuring protein-protein interactions that has also been greatly relied on in this thesis is SPR-based biosensor technology (Biacore). This instrumentation monitors the complex formation and dissociation between macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface that is coated with a ligand molecule. The instrument has been used to investigate numerous different biomolecular systems, including: cytokine growth-factor-receptor recognition (Cunningham et al. 1989; Cunningham and Wells 1989; 1993), coagulation factor assembly (Fan et al. 1998) and virus-cell docking (Rux et al. 1998; Willis et al. 1998). There are several advantageous with using optical biosensors, such as the possibility of real-time measurements allowing 26
for dissection of the interaction into association rate and dissociation rate components, dispensing the need for labeling the biomolecules, Therefore the technique is very useful in the design of antagonists and ligands whose efficacy depends on their binding dynamics (Myszka 1997; Canziani et al. 1999). Sometimes it may not be necessary to conduct a complete kinetic study, but rather an affinity and rate ranking of the analytes, to investigate the relative affinity of two biomolecules to the same target. To facilitate the interpretation it is advantageous if these different binding molecules have approximately the same molecular weights. A number of research groups have shown that using optical biosensor in combination with site-directed mutagenesis provides a powerful technique for identifying structural elements that are key determinants of specificity and affinity (Bass et al. 1991; Albeck and Schreiber 1999; Gårdsvoll et al. 1999).
Bacterial surface receptors Several pathogenic Gram-positive bacteria interact with host proteins through receptors expressed as anchored to the bacterial surface. The biological significance of this phenomenon in relation to pathogenicity and the immunogenic response is not fully clear, but most probably the pathogenic bacteria create a layer of host proteins around themselves (Achari et al. 1992; Sauer-Eriksson et al. 1995; Starovasnik et al. 1996). Surface proteins that are capable of binding to host specific proteins include for example streptococcal protein G (SPG) and staphylococcal protein A (SPA). SPA and SPG have both high affinities for immunoglobulin G (IgG) from different species, and SPG can also bind serum albumin from a variety of species. SPA and SPG have been thoroughly investigated for various biotechnology purposes such as immunological tools in a vast array of immunoassays, and purification of immunoglobulins (Amersham Biosciences 1997). Interestingly, the HSA-binding domains from SPG and the IgG binding domains from SPA share the same scaffold despite the fact that SPG’s IgG binding domain has a different scaffold. The Fc binding parts of SPA and SPG are example of convergent evolution, where two proteins have found two different structural solutions for binding similar proteins with similar affinity.
27
Staphylococcal protein A SPA is a cell-wall associated receptor exposed on the surface of the Gram-positive bacterium Staphylococcus aureus. SPA has high affinity to IgG from various species, for instance human, mouse, rabbit and guinea pig. (Richman et al. 1982; Amersham Biosciences 1997). This indicates that SPA facilitates the ability of the bacteria to also infect animals other than humans. The gene encoding SPA was sequenced by Uhlén and co-workers in 1984 (Uhlén et al. 1984). SPA consists of three different regions; S, being the signal sequence that is processed during secretion (Abrahmsen et al. 1985), five homologous IgG binding domains E, D, A, B and C (Moks et al. 1986) and a cell anchoring region XM (Guss et al. 1984; Schneewind et al. 1995) (see Figure 4). A number of features of the IgG-binding domains on SPA; such as being highly soluble, proteolytically stable, and devoid of cysteins have made these suitable for use as affinity gene fusion partners for production and purification of recombinant proteins (Ståhl 1999). In addition to the most used expression host E. coli, SPA has been successfully expressed also in yeasts, insect cells and mammalian cells (Ståhl and Nygren 1997).
Staphylococcal protein A (SpA) Immunoglobulin binding S
E
D
A
B
C
Z
X
M
57 kDa
7 kDa
Figure 4. The staphylococcal protein A shown here is a cell-wall associated receptor consisting of a signal sequence (S) processed during secretion, five homologous IgG-binding domains (E, D, A, B and C), and a cell-wall attaching structure (XM). Also shown is the commonly used Z domain, corresponding to an engineered version of the B domain of SPA (Nilsson et al. 1987).
28
Each of the five domains in SPA is arranged in an antiparallell three-a-helical bundle of approximately 58 aa. The structure is stabilized via a hydrophobic core. Each domain has high affinity for the Fc part of IgG1, IgG2 and IgG4, estimated to be approximately Kaff =10-8 (M), but shows only weak interaction with IgG3 (Kronvall and Williams 1969; Ankerst et al. 1974; Jendeberg et al. 1997). In addition, each domain has high affinity for the Fab part of certain antibodies (Potter et al. 1996; Jansson et al. 1998). The binding site for the Fc part of the IgG molecule has been shown in a study of the B domain, to involve 11 residues of helix 1 and helix 2 (Deisenhofer 1981). In addition, another study using the D domain showed that the Fab-binding part was located distinctly apart from the Fc-binding part. The 11 residues involved in the Fab interaction are situated on the second and third helices (Graille et al. 2000). Domain B is closest to a hypothetical consensus of the five SPA domains (Moks et al. 1986) and was therefore chosen for further improvements. In order to increase domain B’s own tolerance towards site-specific chemical cleavage of fusion proteins using the chemical cleavage agent hydroxylamine, the sensitive Asn-Gly dipeptide in the B domain at residues 28-29 was changed to AsnAla by site directed mutagenesis (Nilsson et al. 1987), resulting in an engineered domain denoted Z. This allowed for development of bioprocesses where after an initial purification step of ZZ-target fusion proteins with IgG-affinity purification, hydroxylamine could be used to cleave off Z from the target protein via an Asn-Gly dipeptide sequence genetically introduced between ZZ and the target protein. In a second IgG affinity purification step, the cleaved target protein was collected in the flow through, while Z-domain proteins were captured in the column (Moks et al. 1987). While introducing the single mutations A29G to the B domain the Fab interaction diminished, probably due to the fact that the Ala would perturb the interaction between the two molecules (Jansson et al. 1998; Graille et al. 2000). For the application as immobilized affinity ligand for capture of IgG, previous work indicate that a head-to-tail dimer construct of the Z domain has a similar molar binding capacity for IgG as the native SPA molecule, which has five repetitive domains (Ljungquist et al. 1989). The Z scaffold has also been used in protein engineering to introduce new properties. For example 13 surface exposed amino acids at the binding site could be randomized in a phage display library and several new binders were possible to select (Nord et al. 1995; Nord et al. 1997). 29
Streptococcal protein G In 1984 Björck and Kronvall isolated and named the protein G receptor from Streptococcus strain G148 (Björck and Kronvall 1984). SPG is exposed on the surface of the bacteria and consists of a signal peptide that is processed during secretion and repetitive regions capable of binding immunoglobulins and serum albumin from different species, respectively. The albumin-binding region is separated from the immunoglobulin-binding region by a spacer region and a cell-wall anchoring region named W is located in the C-terminus (Olsson et al. 1987) (Figure 5).
Streptococcal protein G Serum albumin binding Ss E A1
B1
BB
ABP ABD
A2
B2
A3 S
Immunoglobulin binding C1 D1 C2 D2 C3
214 aa
W
63 kDa
25 kDa
121 aa
15 kDa
46 aa
5 kDa
Figure 5. Schematic presentation of SPG. The albumin and IgG- binding regions consist of three albumin and IgG-binding domains respectively (Olsson et al 1987), with a spacer region, S between these two regions. The signal peptide Ss is processed during secretion and W is the cell-anchoring region. The albumin-binding region has been cloned in different constructions denoted BB, ABP and ABD containing 2.5, 2 and 1 domains respectively.
HSA binding region The HSA-binding region of SPG consists of three homologous albumin-binding domains (ABDs) separated by linkers of approximately 30 residues each (Olsson et al. 1987). Different parts of the albumin-binding region have been cloned and characterized, (BB, ABP and ABD) with 2.5, 2 and 1 domains respectively (Ståhl and Nygren 1997) (Figure 5). Each ABD domain consists of approximately 46 amino acids. The domain has no additional stabilizing features such as bound ligands, metal ions or disulphide bridges (Kraulis et al. 1996). HSA is postulated to contain one binding site for protein G, formed by loops 6-8 (Falkenberg et al. 1992). Protein G
30
shows strong interaction with serum albumin from different species including rat, mouse, rabbit and human, but very low interaction to bovine serum albumin (Nygren et al. 1990; Falkenberg et al. 1992; Johansson et al. 2002). These results imply that streptococcus strain 148 could also be pathogen for rat and mouse. Many of the positive features associated with the Z-domain; for example, stability to proteolysis, high-level production, ability to be secreted, and a solubilization of the fused protein can also be related to ABD (Nygren et al. 1988; Ståhl et al. 1997). The ability of the ABD to bind serum albumin is also of interest from a therapeutic perspective. Various difficulties have been encountered when administering proteins for therapy purposes, such as low efficacy due to a rapid clearance rate from the blood. Because kidneys generally filter out molecules below 60 kDa, efforts to reduce clearance rates have focused on increased molecular size through glycosylation or the addition of polyethylene glycol polymers (PEG) (Dennis et al. 2002). Alternatively, the HSA binding ABD has been investigated as a gene fusion partner to produce ABD-tagged proteins capable of binding to circulating serum albumin after administration. Serum albumin is a protein with a long half-life exemplified by HSA, which has an in vivo T1/2 of 19 days in humans (Peters 1985) Hence the half-life of ABD would be extended by the binding to serum albumin. By fusing the human soluble complement receptor type 1 (sCR1) to the albumin binding part of the SPG Makrides with co-workers were able to an extended half-life of the sCR1 in rats (Makrides et al. 1996). Furthermore, the albumin-binding region BB seems to have immunopotentiating properties when genetically fused to an immunogen (Power et al. 1997; Sjölander et al. 1997; Libon et al. 1999). This property has not been fully elucidated yet, but it has been proposed that it might result from T-cell epitopes (Goetsch et al. 2003) or the serum albumin-binding affinity (Makrides et al. 1996; Sjölander et al. 1997). In a recent study, it was proposed that the region involved in serum albumin binding also contained a T cell epitope (Goetsch et al. 2003).
31
IgG binding region The IgG-binding region of SPG consists of three individual IgG-binding domains, where each one has high affinity for the Fc and the Fab regions of IgG. In contrast to protein A, SPG exhibits binding to all human subclasses of IgG, including IgG3. SPG also has a high affinity for IgG from several animals, including mouse, rabbit and sheep (Åkerström et al. 1985; Amersham Biosciences 1997). The domains consist of about 55 amino acids and the structure is constituted of two b-hairpins that are associated to form a four stranded mixed antiparallel/parallel b-sheet with a single ahelix lying across one face of the sheet. No extra stabilizing features such as metal ions or disulfide bridges is needed for the stability of the domains as in the case of the three helical scaffold of SPG and SPA (Åkerström et al. 1985; Gronenborn et al. 1991). Interestingly, the IgG binding domains of SPG show clear structural similarities with the immunoglobulin light chain binding protein L from Peptostreptococcus magnus (Wikström et al. 1994), despite the fact that their sequence homology is low. The overall folds of proteins L and G are similar, though the two proteins also demonstrate significant differences, the orientation of the ahelices in protein L run nearly parallel to the b-sheet, while the helices in protein G run diagonally across the b-sheet (Wikström et al. 1995). The Fc binding part of the SPG molecule is located mainly in charged and polar residues of the helix and the loop connecting the helix with the third b-strand (Gronenborn and Clore 1993). Protein G binds Fc at the region that connects the CH2 and CH3 domains, thus SPA and SPG bind to overlapping sites of the Fc-molecule. Interestingly, the Fab binding region is almost non-overlapping with the Fc binding part of the molecule, located mainly in the second b-strand and in the loop between the first and second b-strand (Lian et al. 1994). In summary, the bacterial domains originating from the surface receptors of SPA and SPG are small, easy to produce, stable, and can be secreted. In addition, several studies have shown that it is possible to change a number of amino acids at the surface and retain the native structure (Klemba et al. 1995; Nord et al. 1995; Gräslund et al. 2000). These features make the domains very interesting for protein engineering.
32
Affinity chromatography purification A number of different protein purification techniques exist today, including ionexchange chromatography, reverse-phase and gel filtration, which take advantage of differences in electrostatic characteristics, hydrophicity or the size of proteins in order to separate them. One very powerful technique for protein purification is based on biospecific interactions, namely affinity purification. A number of different interactions have proven successful in this area, including for example interactions between enzyme and substrate, bacterial receptor and serum protein, and antigen and antibody (Nilsson et al. 1997). Some of the most commonly used affinity partners have been listed in Table 1. Table 1. Commonly used affinity fusion systems, modified from Nilsson et al. 1997.
Affinity tag
Ligand
Elution
References
Protein A Z Albumin binding protein Glutathione S-tranferase (ABP) Polyhistidine tag (GST) Maltose binding protein FLAG peptide
hIgG hIgG HSA Glutathione
low pH low pH low pH Reduced Imidazole/low glutathione Maltose pH EDTA/low pH
Ståhl and Nygren. 1997 Nilsson et al. 1987 Nygren et al. 1988 Smith and Johnson 1988 Porath et al. 1975 di Guan et al. 1988 Hopp 1988
+
Me2 /Chelator Amylose mAb1, mAb2
These different systems all have various characteristics, which have to be taken into consideration when selecting purification strategy. Common for all listed systems is that the target protein needs to be modified to include the affinity gene fusion partner, which, if a native target product is desired, calls for efficient means to release the affinity tag after the purification. In other and more attractive formats of affinity chromatography, native target proteins can be directly purified, which obviously requires that ligands recognizing such native targets are available. By using combinatorial chemistry, it is possible to generate new specific binders that could be used as affinity ligands (Nord et al. 1997). Affinity chromatography has a number of advantages over other purification techniques currently available; for instance it is an easy, fast and selective means of capturing biomolecules. This allows the introduction of an affinity-purification step early in the purification chain, and the number of successive unit operations can be reduced, which would be a benefit in industrial purification (Harakas 1994; MacLennan 1995). Achieving the final product in largescale industrial applications often requires the overall purification procedure to include several consecutive unit operations. Previously it has been shown to be 33
advantageous to conduct initial purification before the affinity chromatography step, for example precipitation or ion-exchange chromatography (Cutler 1996) for removing some of the major contaminants and thereby increasing the column lifetime. Purification of plasma proteins, such as human serum albumin (HSA) or Immunoglobulin (IgG) has for a long time been employed for therapeutic applications. In the mid-1940s, ethanol fractionation was introduced and is still in use today together with other separations techniques, such as ion-exchange and affinity chromatography (Foster 1992). Recently, affinity purification has been applied in fine and rapid capture of plasma proteins in large-scale production. Some of the proteins produced and purified in this process include factors VIII, IX and XI, von Willebrand factor, protein C and antithrombin III (Burnouf and Radosevich 2001). Using affinity techniques has dramatically improved the purity of the products, and thus unwanted side effects, such as hemolysis, hypotension and fever is considered to be very rare (Burnouf and Radosevich 2001). Development of affinity chromatography for purification of plasma products in large-scale production faces a number of different issues, such as the selection and development of ligands, leaching problems, the stability of ligands exposed to cleaning procedures, and the capacity of the column (Burnouf and Radosevich 2001). In an industrial plant, the cleaning in place CIP solution is routinely applied throughout the process after each purification procedure. This is done to diminish contamination between different processes or batches. A universal chemical agent that has been shown to be successful in the inactivation of most microorganisms, such as bacteria, viruses, yeasts and also destroys endotoxins is NaOH in solution (Girot et al. 1990; Burgoyne et al. 1993; Asplund et al. 2000). However, the harsh conditions associated with such CIP procedures involving high pH will decrease the performance of many proteinaceous ligands in an affinity-chromatography process. The most sensitive amino acids to alkaline treatments in several studies have been shown to be Asn and Glu (Geiger and Clarke 1987; Kossiakoff 1988; Lura and Schirch 1988). The overall stability of the protein is also dependent on secondary structure elements surrounding these amino acids and neighboring amino acids (Robinson and Robinson 1991; Wright 1991; Kosky et al. 1999; Xie et al. 2000; Robinson and Robinson 2001). A more detailed description about the susceptibly of asparagine residues in alkaline conditions can be found in the section of the thesis entitled tolerance to alkaline condition. Improving the stability of a protein in alkaline treatment will not 34
only improve its performance as an industrial affinity purification ligand, it might also decrease the leaching problem. As a result, the costs of using a proteinaceous ligand in a large-scale purification scheme will be reduced. The use of affinity-based chromatography has as a consequence of the above issues, hitherto not been as extensively used as predicted.
35
Present Investigation The research presented in this thesis covers a number of different applications of protein engineering technology where site-directed mutagenesis has been used to study and improve the performance of bacterial domains in biotechnological applications. In order to explore the binding surface of ABD originating from SPG, a number of surface exposed amino acids were substituted and the different mutants were analyzed with SPR technology. Thereby, the contribution from the different amino acid on the affinity to HSA was discovered. To improve the ABD for biotechnology applications, amino acids sensitive to alkaline conditions were replaced to obtain an increased tolerance to alkaline conditions. In addition, different connective linkers were analyzed, for the production of divalent ligands. Since the stabilization strategy worked successfully in improving the alkaline stability of ABD, a similar strategy was investigated for alkaline stabilization of two other protein domains, namely the IgG-binding C2 domain originating from SPG and the Z domain originating from SPA.
HSA binding domain (I) Mutational analysis of an albumin binding domain An alanine scanning procedure was carried out to localize the surface on ABD responsible for the interaction with HSA. Initially, residues in positions E3, Y20, E32 and E40 were replaced. These amino acids were chosen as they were surface-exposed, located on all three a-helices, and pointing in different directions. In order to ensure an easy purification procedure, all ABD constructs were fused to the IgG- binding Zdomain. Of the four first residues chosen, Y20A was determined to contribute most to the interaction (see Table 2). To determine more precisely which amino acids were involved in the interaction, further alanine substitutions were made in helix 2. Residues in positions S18, D19, Y21, K22, N23, L24 and K29 were replaced by alanine to determine their respective contributions to the interaction with HSA. To analyze the effect of multiple mutations on structure and affinity, three additional mutants were made, ABD(Y20A, E40A), ABD(Y20A, E32A) and ABD(S18, Y20A,
36
K22A). An extensive biosensor-based binding study was performed to analyze the affinity of the different mutated protein domains to HSA. The CD spectra of the mutated proteins were recorded to analyze the effect on the secondary structure, as this has proven to be suitable for detecting structural changes in a-helical proteins (Johnson 1990; Nord et al. 1995). The various mutants of ABD were subjected to a subtractive CD spectroscopic analysis in which the signal contribution from the Z domain was subtracted (Nord et al. 1997). The results from the CD measurements show that the backbone configuration is similar for all constructs. However, because the exact structures of the different mutants are unknown, minor changes in the structure due to the mutations cannot be ruled out (Clackson et al. 1998; Vaughan et al. 1999). Table 2 presents the results of the kinetic study with the SPR technology (Biacore). Residues in the second helix give a larger contribution than residues in the first and third helices. In the same table, it can be seen that the two tyrosines at positions Y20 and Y21 located in the second helix, are the residues that have the largest effect on the affinity between ABD and HSA. These two tyrosines are both located in the Nterminal part of the second helix, and are also surface-exposed. Interestingly, residue D19, which is close to both tyrosines, does not appear to contribute to the interaction. Both HSA and ABD are negatively charged at the pH used in the binding studies. This can explain why replacing a positively charged Lys with an Ala in position 29 decreased the association rate, and accordingly why replacing the negatively charged Glu acids in positions 32 and 40 with an Ala increased the association rate. In order to further verify the position of the binding surface, three mutants were constructed through changing three neighbouring amino acids at a time. Alanine substitutions were made in helices one and three, in positions where the amino acids were pointing away from the postulated binding site. The resulting proteins were: ABD*(E3A, V6A, L7A), ABD*(R10A, E11A, K14A) and ABD*(K35A, I38A, D39A). More information about ABD* can be found in the section of the thesis entitled Stabilization of ABD towards alkaline conditions. All three mutants were shown to have similar or better affinity to HSA than the parental molecule. Hence, the conclusion is that the main part of the binding site is located in the second helix, and the two tyrosines have the greatest impact on the stability of the interaction between ABD and HSA. Interestingly, Johansson and co-workers found in an NMR study that a larger part of the ABD molecule was found to be affected while binding to HSA 37
(Johansson et al. 2002). This could be explained by the fact that in the NMR experiment, interaction surfaces described by chemical shift perturbation methods are usually larger than the region in direct contact (Spitzfaden et al. 1992; Foster et al. 1998). This illustrates the differences between using these two methods and how they could complement each other. Table 2. An overview of the kinetic study of the ABD variants carried out on the Biacore.
koff -3
kon -1
5
-1 -1
ZABD variant
[10 s ]
[10 M s ]
wt E3A S18A D19A Y20A Y21A K22A N23A L24A K29A E32A E40A Y20E32A Y20E40A S18Y20K22A
0.6 (0.2) 0.6 (0.3) 3.5 (1.6) 0.8 (0.3) 5.1 (2.1) 3.9 (2.4) 1.3 (0.7) 0.9 (0.3) 0.6 (0.4) 1.1 (0.4) 2.4 (0.9) 1.2 (0.5) 17 (11) 6.8 (2.1) 6
1.4 (0.2) 1.4 (0.7) 1.3 (0.3) 1.5 (0.3) 0.9 (0.2) 0.1 (0.07) 0.7 (0.2) 1.0 (0.1) 0.3 (0.02) 0.3 (0.1) 2.5 (0.3) 2.6 (1.5) 0.5 (0.2) 1.4 (0.1) 0.0001
Kaff 7
DDG -1
[10 M ] 25.9 22.2 3.9 20.9 1.9 0.3 5.6 12.7 4.5 3.4 12.2 29.5 0.5 2.1 0.0002
[vs wt] [kcal/mol] 0.0 (0.2) 0.1 (0.1) 1.1 (0.1) 0.1 (0.3) 1.5 (0.2) 2.6 (0.2) 0.9 (0.2) 0.4 (0.1) 1.0 (0.2) 1.2 (0.6) 0.4 (0.3) -0.1 (0.6) 2.3 (0.1) 1.5 (0.1) 7
Standard deviations are given in brackets. Note that as the very low affinity of the triple mutant leads to problems in the detection of the binding, no standard deviation is given for that specific mutant.
Electrostatic interactions have been found to be an important tool for the association of biomolecules (Selzer and Schreiber 1999; Selzer et al. 2000; Schreiber 2002). This could also be seen in the interaction between ABD and HSA. Some of the mutations that seem to affect the association could be explained by removal or addition of charges, for example E32A and E40A. Interestingly, when exchanging a glutamatic acid for an alanine in position 32, both the association rate and dissociation rate increase (see figure 6). It has been shown in previous studies that a mutation can have a specific effect on either kon or koff (Schreiber and Fersht 1995) giving an unchanged 38
affinity constant despite large changes in the on- and off-rate. In the third helix, substitution both in position 32 and 40 results in a slightly increased kon. The importance of the electrostatic interactions could also be seen when alanines were introduced into the three different ABD* variants where the side chains are located on the opposite side of the postulated binding site. The binding analyses show that the off-rate is almost identical for all three mutants; indicating that the stability of the ABD-HSA complex has not been affected. However, one of the mutants shows an increased on-rate. This can be explained by the change of charge of the molecule resulting from changing negatively charged aspartic acid to an uncharged alanine, which might increase the electrostatic attraction between HSA and ABD, as both are negatively charged at the pH used in the binding studies. Furthermore, in this triple mutant, a positively charged lysine was also exchanged for an alanine. This might help to direct the HSA-binding surface of ABD to the HSA molecule. These two mutations could thereby increase the “steering region”, as described in the paper by Seltzer and co-researchers (Selzer et al. 2000). The steering volume is defined as the volume surrounding a molecule in which it through electrostatic interactions could change its movement from random diffusion to directional movement towards the complex formation.
39
A
B
Figure 6. The three-dimensional structure of ABD displaying the effect of different mutations, where: (A) shows the change in kon by mutation, with orange indicating a decreased on-rate, green an increased on-rate, and yellow an unchanged on-rate where changing a residue to alanine; (B) shows the change in the off-rate when replacing certain residues for alanine, with orange indicating that the off-rate increases upon mutation and yellow that the exchange to alanine does not affect the koff
Stabilization of ABD towards alkaline conditions (II & III) A chemically stable protein ligand with high affinity for albumin would be interesting from a biotechnological point of view, due to the fact that HSA is the most abundant protein in clinical use worldwide. HSA is used to restore colloidal osmotic pressure in severe burn damages, and to treat the of loss of albumin in traumatic accidents. HSA for industrial purposes is produced by fractionation from serum, though a recombinant process in yeast is also available (Quirk et al. 1989). The work presented in Papers II and III includes a protein engineering strategy to stabilize and optimize proteinaceous ligands for large-scale applications, namely ABD. The model chosen for this thesis was an ABD domain originating from SPG (Kraulis et al. 1996). A more detailed description of this domain can be found in the chapter of the thesis entitled Bacterial surface receptors. The asparagine residues that are known to be sensitive to alkaline conditions (Geiger and Clarke 1987; Kossiakoff 1988) were replaced by comparing the ABD-domain to homologous sequences (see Figure 7), i.e other albumin binding domains. This procedure suggested that, asparagines in positions 9 and 27 could be replaced by leucine and lysine, while
40
asparagine in positions 23 and 26 could be replaced with aspartic acids. The new mutant ABD (N9L,23D,26K,27D) was denoted ABD*. The single domain was then characterized with respect to its affinity, stability and function as an affinity ligand.
LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP --K--AD-LK-FN----------------------D-QAQVVESAK --------------------H-----K------IME-QAQVVES -DN--NA-LK-F-R------------K------IME-QAQVVES -S---EM-I----AN----F--DK-DD-------V--K-L--NS --------L-------------D--DK-------------------
ABDwt SPG ABD-1 SPG ABD-2 DG12 ABD-1 DG12 ABD-2 ABD*
Figure 7. The primary sequence of ABD (here denoted ABDwt) (Kraulis et al. 1996) and ABD*. Shown are also homologous sequences of albumin binding domains derived from other strains of Streptococcus as well as the homologous domains from SPG. These homologous sequences were referred to when constructing ABD*. SPG is derived from Streptococcus G148 (Olsson et al. 1987), DG12 from Streptococcus DG12 (Sjöbring 1992) and PAB (ALB-strains) from Peptostreptococcus magnus (de Chateau and Björck 1994).
After substituting amino acids in a protein scaffold, it is important to characterize how the mutations affect the protein. A kinetic study was carried out with the Biacore, and the structure along with the thermal and chemical stabilities were analyzed with CD. The kinetic parameters for binding ABDwt and ABD* to HSA is shown in Table 3. The binding behavior of the mutated protein was similar to the binding behavior of the native protein. The dissociation rate constant was almost identical, while a decrease could be seen in the association rate constant. An explanation could be that the introduced mutations affect the “steering region”, as has been shown by Selzer and co-workers and is discussed in the chapter of the thesis entitled Protein-protein interactions (Selzer et al. 2000; Selzer and Schreiber 2001).
41
ABD ABD*
ka
kd
KA
DDG
[M-1 s-1]
[s-1]
[M-1]
[kcal mol-1]
11x104
1.2x10-3
9.0x107
-3
7
2.5x10
4
1.3x10
1.9x10
0.9
Table 3. Parameters of the binding of ABD and ABD* to HSA. ka is an association rate constant. kd is a dissociation rate constant. KA is the affinity constant. DDG is the difference in binding free energy between ABDwt and ABD*.
The ABD* molecule also appears to have a similar secondary structure to ABDwt according to the CD measurement. However the a-helical content seems a little decreased in ABD* compared to ABDwt. The thermal and chemical stabilities were analyzed with a CD spectropolarimeter. Both proteins show high stability to both types of denaturation and consequently a defined unfolded state was hard to reach during the measurements (see Figure 8). Therefore the midpoint values of temperature or denaturant-induced transition have been used as estimations for melting points. The Cm value for ABDwt, when denaturating with GdnHCl, was estimated to be 4.2M, and more than 5 M for ABD*. When analyzing thermal stability, the mutant was found to be more stable than the native molecule. The Tm value was estimated to be 70 ºC for ABDwt , and over 80 ºC for ABD*.
42
Figure 8. CD-analysis of ABD and ABD*, for a comparison of the structural stability. The protein concentration was 0.5 mg ml-1 in a phosphate buffer of pH 7. The signals at 222 nm have been transformed to MRE. (A) Stability towards chemical denaturation. The temperature is 20°C and concentration of chemical denaturant, GdnHCl, is varied between 2 and 6 M. (B) Stability towards thermal denaturation. The temperature was varied between 40 and 95ºC.
Stability to alkaline treatment was analyzed by three different approaches. In order to analyze the functionality and stability of ABD* the protein was immobilized to a standard affinity matrix by N-hydroxysuccinimide (NHS) amine coupling chemistry. An analysis of the capacity of the new column was done by loading an excess of HSA into the column then measuring the amount of eluted material to determine the total capacity.
43
The selectivity of the mutated protein was analyzed and compared with the wild type by mixing the HSA with protein extract from E. coli. A washing step was included between each purification cycle, wherein a solution containing 0.5 M NaOH was pumped through the column for two minutes. After 34 minutes of exposure to high pH, the ABDwt column showed a 50% decrease in capacity, while the ABD* column on the other hand, seemed to retain almost all capacity despite the harsh treatment. After rounds 2, 8 and 15, the eluted material was analyzed with SDS-PAGE and compared with pure HSA to examine how the specificity had been affected (see Figure 9). The results indicated that the column with immobilized ABD* retained its HSA selectivity. 1
2
3
4
5
6
7
8
kDa 94 67 43 30 20 14
Figure 9. Analysis of different protein fractions after affinity purification with an integrated CIP-step using ABD* as affinity ligand coupled to the matrix. SDS-PAGE gel (4-20% gradient) under reducing conditions. Lane 1 and 2, ABD and ABD* respectively; lane 3, a molecular weight standard; lane 4, E. coli disintegrate spiked with HSA before column loading. The purification procedure was repeated several times; lane 5, 6 and 7, show the eluted material after round 2, 8 and 15 respectively; lane 8 shows HSA reference.
After the work with the single domain was done, an optimization approach was undertaken to increase the capacity for HSA. In order to improve the ABD* domain as an affinity ligand in HSA purification, a head-to-tail dimeric version was constructed through genetic engineering. A divalent capturing ligand could potentially contribute here by creating advantageous avidity effects in the binding between the ligand and the target, possible binding of two HSA molecules, as well as by providing a spacer between the solid phase and the solution. Hence, a larger ligand would have a beneficial effect on the presentation of the ligand to the surrounding solution. The 44
divalent ABD* motif was also equipped with a unique C-terminal cysteine residue, providing a tool for directed coupling of the ligands to the solid chromatographic resins. Previous work done on linker regions connecting domains has shown that these regions are important for the stability and folding of the protein (Robinson and Sauer 1998) and careful consideration has to be taken when designing them (Alfthan et al. 1995; Arai et al. 2001; George and Heringa 2002). These regions are highly flexible and therefore susceptible towards proteases (Hubbard 1998) and CIP conditions (Kosky et al. 1999). Three different linkers were investigated, VDANS, VDADS and GGGSG (each given a single amino acid letter code), referred to as A, B and C respectively. The first linker (A) was a result of the cloning procedure. In the second linker (B), the asparagine was replaced by an aspartic acid according to the previously reported fragility of Asn-Ser sequences (Geiger and Clark 1987). The third linker (C) was composed of glycine and serine residues that in previous reports have been found to be stable and highly flexible (Argos 1990; Alfthan et al. 1995; Takeda et al. 2001). The length of these designed linkers are about five residues, which can be compared to the naturally occurring linkers in SPG, which are about 15 to 30 residues (Olsson et al. 1987). On the other hand, in SPA the linkers between the domains are approximately 8 to 13 amino acids (Uhlén et al. 1984). Binding studies were performed to elucidate differences in the binding capacity between the monovalent original construct ABD* and the divalent variant, the ABD* dimer A. Additionally, two different chemistries were used to examine the importance of the coupling-chemistry when attaching the domains to a solid phase. The nonspecific amine coupling was compared to directed immobilization through the Cterminal cysteine. As can be seen in Figure 10, the directed coupling gives a higher overall binding capacity than when non-directed chemistry was used. The divalent variant shows a higher proportion of active domains than the monomer does, when using non-direct coupling chemistry.
Interestingly using a directed-coupling
chemistry, the divalent variant shows the same proportion of active domains as the monovalent variant.
45
Responsnse [RU] 800
700
600
ABD*dimerA
500
400
ABD*monomer
300
200
100
0 0
200
400
600
800
100 0
120 0
140 0
Time [sec]
Response [RU] 1600
[RU ]
1400
1200
ABD*dimerA ABD*monomer
1000
800
600
400 200
0 0
200
400
600
800
1000
1200
1400
Time [sec]
Figure 10. Biosensor analysis of the interactions between injected HSA and different ABD constructs. The monovalent ABD* and one divalent variant (ABD*dimerA) were immobilized on the chip surface by (A) NHS-chemistry (non-directed) or (B) thiol-chemistry (directed), respectively. The amount immobilized ABD*-domains was similar in all cases (for NHSchemistry: ABD* 970 RU, and ABD*dimerA 900 RU; for thiol-chemistry: ABD* 905 RU, and ABD*dimerA 894 RU). A 300 nM HSA solution was injected over the different surfaces. Mean values from duplicate samples are shown.
The different divalent ABD* variants as well as the monovalent ABD* domain were coupled separately to activated sepharose by recruiting their C-terminal cysteines, resulting in directed coupling. The different affinity media were packed into columns and used in affinity purification experiments for HSA-binding capacity studies. The results have been summarized in Table 4. These results were in agreement with the biosensor studies that the divalent and monovalent proteins were equally accessible for HSA molecules when directed-coupling chemistry was used for immobilization. Furthermore, the data indicate that it might be possibly to increase the capacity of an 46
affinity column by immobilizing a divalent ligand to increase the amount of coupled ligands on the surface of the matrix. In the experiments conducted here, the ABD*dimerB (13 mg) exhibits more than twice the capacity of the monovalent variant ABD* (5 mg). Table 4. Dynamic binding capacity of the different ABD* chromatography matrices.
Construct
Ligand density [mg/ml]
Column volume [ml]
Coupled ABD*domain [nmol]
HSA [nmol]
Bound HSA/ coupled ligand
HSA [mg/ml]**
ABD* ABD*dimerA ABD*dimerB ABD*dimerC
3.0 3.4 5.1 2.6
1.06 1.00 0.82 0.98
440 534 656 406
71 95 159 86
0.16 0.18 0.24 0.21
5 6 13 6
Mw for ABD* and ABD*dimer A, B, and C are 7221.3, 12753.7, 12051.7, and 12585.7 respectively. ** The values have been weighed to the total column volume, i.e. capacity of the column/volume of the column.
Further, the different affinity media with their attached monovalent and divalent affinity ligands were subjected to a stability study. The results of this study have been summarized in Figure 11. All the ligands possess a high tolerance to alkaline exposure, however the ligands containing the VDADS linker show the highest tolerance. Even after as long exposure as 7 h in 0.1 M NaOH, the capacity of the ABD*dimerB remained at about 95% compared to approximately 85% for all the other ligands. It is interesting that a divalent ligand could be more tolerant to alkaline conditions than the monovalent ABD* ligand. The reason for this remains elusive, but it can be speculated that the two domains in a divalent ligand could exert a mutual stabilizing effect relating to refolding after exposure to alkaline conditions (Robinson and Sauer 1998; Wenk et al. 1998).
47
Capacity [%] 100,0
Linker B Linker A Linker C ABDmonomer
80,0
60,0
40,0
20,0
0,0 0
50
100
150
200
250
300
350
400
450
Time of exposure [min]
Figure 11. Influence of alkaline sanitation on the HSA binding capacity of an affinity column. The monovalent ABD* ligand and the three divalent ABD* ligands were separately analyzed in a column chromatography protocol in order to explore the tolerance to repeated alkaline sanitization (0.1 M NaOH). After injection of HSA, the captured protein were eluted and the binding capacity determined. In order to clean the affinity purification device, 0.1 M NaOH was pumped though the column for 20 minutes between each purification round. The ligands were immobilized using thioether chemistry by taking advantage of the C-terminal cysteine. ◊;ABD*dimerA, o; ABD*dimerB, D; ABD*dimerC, x; ABD*monomer.
48
IgG binding domains Stabilization of C2 towards alkaline conditions (IV) The same strategy that was applied to enhance ABD for industrial applications was investigated for alkaline stabilization of the IgG-binding domain C2 originating from SPG. The sensitivity of these domains to alkaline pH has earlier been reported (Goward et al. 1991). Although this structure differs considerably from the ABDdomain worked with earlier, the same stabilizing strategy could possibly still be applied. First, the sensitive Asn residues were replaced and then a divalent variant was constructed in order to increase the capacity of an affinity matrix for IgG capture. Instead of replacing all the asparagines simultaneously, each asparagine was replaced one by one, to investigate their contribution to the deactivation during alkaline treatment (Stephenson and Clarke 1989; Wright 1991). In addition, the aspartates and glutamines were replaced to investigate whether the tolerance to alkaline conditions could be increased even further (Stephenson and Clarke 1989; Wright 1991; Tomizawa et al. 1995). The amino acid composition of C2 is shown in figure 12, with the sensitive asparagines highlighted. Three single mutants were designed to resolve the contribution of each asparagine to the deactivation rate, C2N7A ,C2 N34A and C2N36A . In addition, one variant was constructed with two asparagines exchanged for alanine (denoted C2N7,36A), and one constructed with three asparagines exchanged for alanine (denoted C2N7,34,36A). A second generation of mutants was then generated using the most stable mutated variant (C2N7,36A) as a scaffold. Two single mutants and one triple mutant were designed yielding a protein where the aspartate residues were substituted for glutamate residues in order to retain the overall charge, and these were designated: C2N7,36AD35E, C2 N7,36AD39E and C2N7,36AD21,45,46E respectively . In order to explore the sensitivity of the single glutamine in the structure, C2N7,36A was used as a scaffold again and the glutamine was substituted for alanine resulting in C2N7,36AQ31A.
49
TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
A
A A
Figure 12. The 55-residue sequence of the C2 domain (Olsson et al. 1987). The aspargagine were replaced with alanine.
The different mutants of C2 were analyzed regarding affinity, structure and stability using Biacore, CD and isoelectric focusing gel electrophoresis (IEF). In addition, an affinity chromatography scheme was used to analyze their tolerance to alkaline treatment. The strategy involved the replacement of each asparagine residue on a one-by-one basis to investigate and assess each individual asparagine’s contribution to function and stability as an affinity ligand. Mutations N7A and N36A were found to be optimal for increasing the tolerance of the IgG-binding domain C2 to alkaline treatment. In addition, genetically fused dimers of this double mutant were designed and exhibited same tolerance as the monovalent double mutant. SPR technology was used to investigate the interaction between the Fc fragment of IgG and the C2 variants. As can be seen in Table 5, C2, C2N7A, C2N36A and C2N7,36A all had approximately the same kinetic properties. Table 5. Parameters of the interaction of C2 and C2 mutants to Fc from human IgG, measured by SPR. All calculations have been done with Fc immobilized on the surface.
C2 C2N7A C2N36A C2N7,36A
ka [M-1 s-1]
kd [s-1]
KA [M-1]
2.9x105 2.3x105 2.0x105 2.2x105
27.0x10-3 21.1x10-3 13.2x10-3 13.4x10-3
1.1x107 1.1x107 1.5x107 1.7x107
ka is the association rate constant, kd is the dissociation rate constant and KA is the affinity constant (ka/kd).
Potentially, a divalent ligand also could beside the availability of two affinity sites lead to advantageous avidity effects in the binding between the ligand and the target. It could also provide a beneficial effect by effectively presenting the immobilized ligand, by providing a spacer. 50
A CD study was carried out to investigate the structural impact the different mutations had. These CD results indicated that all variants exhibited similar secondary structure contents as the parental protein G fold, although the CD signal was slightly decreased for some of the variants compared with the wild-type C2, indicating that only small changes arose in the secondary structure on mutation. The variant C2N7,36A and the wild-type C2 were subjected to thermal and chemical denaturation with GdnHCl in order to analyze their structural stabilities. The change in signal upon denaturation was measured at 217 nm (a typical minima for a b-sheet proteins). In both studies, all proteins adopted clearly defined pre and post transitions, and subsequently the signals could be transformed to Fapp. For the wild-type C2, the thermal Tm1/2 was 77 ºC and Cm1/2 was 2.9 M; The mutant C2N7,36A had a slightly lower Tm1/2 of 71 ºC, and also a lower Cm1/2 of 2.5 M. As has been shown elsewhere (Geiger and Clark 1987; Tyler-Cross and Schirch 1991) the Asn residues can be modified in alkaline conditions to Asp or isoAsp. This results in an increased negative charge on the protein, which can be studied by isoelectric focusing (IEF). The IEF study indeed showed that after two hours of exposure in 0.1M NaOH, the C2 protein undergoes a transformation to a new more negatively charged species. The double mutant on the other hand revealed no additional charge indicating that the deamidation-susceptible residues were substituted. The ability of C2 and all mutated-C2-variants to function as affinity ligands in an ordinary purification scheme for IgG antibodies was investigated by immobilization of the different domains on HiTrap‘ columns (Amersham Biosciences). Between the different cycles, a two-minute CIP step consisting of 0.1 M NaOH was integrated. The results of the capacity study presented in figure 13 show that after a total of 18 minutes of exposure to NaOH, the C2 column had only about 30% capacity left. The C2N7A column showed a similar loss of binding capacity, while the C2N34A column lost capacity even faster. The C2N36A column showed a significantly higher stability; as did the double mutant C2N7,N36A column. In an attempt to increase the stability even further, several mutants were designed in which the electrostatic nature of the domain was intentionally retained through replacement of aspartate with a glutamate. The double mutant C2N7,36A was used as a scaffold for introducing an alanine to the single glutamine residue substitution. However, the different aspartate and glutamine
51
substitutions revealed no further tolerance to the CIP treatment (see Figure 13). In conclusion, it was possible to stabilize one of the IgG-binding domains from streptococcal protein G in respect to alkaline conditions, by employing a protein engineering strategy. Capacity [%] 120 100
C2N7,36Adim C2N36A C2N7,36A C2N7,36AD35E C2N736AD39E C2N7,36AD21,45,46E C2N7,36AQ31A
80 60 40
C2 C2N7A
20
C2dim
C2N34A
0 2
4
6
8
10
12
14
16
18
20
22
24
26
28
Time of exposure [min] Figure 13. Comparison of capacity after repeated CIP-treatment following a standard affinity chromatography protocol on C2 and all the mutated variants C2-variants as immobilized ligands. The cleaning agent was 0.1 M sodium hydroxide.
52
Stabilization of Z towards alkaline conditions (V) SPA is one of the most widely used affinity ligands in affinity chromatography. Previous studies have shown that SPA-based chromatography media are relatively stable in CIP conditions. A loss of about 1% in binding capacity with exposure to 0.5 M NaOH for 15 minutes has been reported (Hale et al. 1994). However for large-scale applications it would it be valuable to have an even more stable ligand. As a starting scaffold for further stabilization, the Z domain originating from the B domain in SPA was used. The three-dimensional structure of the Z domain has been illustrated in Figure 14, indicating all the asparagines and the different substitutions made in this study. The Z domain consists of 58 amino acids, which includes eight asparagines in positions: 3, 6, 11, 21, 23, 28, 43 and 52 (Nilsson et al. 1987).
N3
N6A
N11S
N28A
N43E
N23T
N52A N21A
Figure 14. The three-dimensional structure of the Z domain (Tashiro et al. 1997). All asparagines and the different substitutions are indicated.
In an initial trial, phage display technology was used as a means to select Z variants with retained IgG binding capacity after substitutions. The library was designed with randomizations at positions 21, 29 and 52. Position 29 was chosen to investigate whether it was possible to retain the Fab interaction with an increased alkaline stability. The selection procedure consisted of three rounds of panning with IgG as bait. After the selection, a number of colonies were sequenced and analyzed. Since 53
the Z-domain already is significantly stable to alkaline conditions it was not possible to introduce alkaline treatment in the selection procedure. Therefore none of the selected binders achieved an increased alkaline stability compared to the native Z domain. An alternative strategy to phage display is to use a by-pass mutagenesis method (Kotsuka et al. 1996). This strategy involves the use of a domain analogue as starting template, into which a destabilizing mutation has been introduced to simplify the search for variants of increased stability. Secondary mutations are then introduced and the effects analyzed. Mutations that improve stability of the destabilized molecule are then grafted into the original protein. Previous studies on the Z domain have shown that a mutation of phenylalanine 30 to an alanine, destabilize the structure about 3.5 kcal/mol (Cedergren et al. 1993). The original phenylalanines side chain contributes to the stability by being part of the hydrophobic core.
Z MKAIFVL GTAD Z(F30) -------NAQHDEAVD E D A B C
NKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK ---------------------------A----------------------------
AQHDEA------QV-NM----AD---G--------------V-G--Q----S---ADAQQ-N---D--S------NM---------G----------------G------ES---AD -N-------------NM---------G----------------S------ES---AD --------------------------G----------------------------AD --------------------T-----G----------V-KEI--------------
Figure 15. Amino acid alignment of the Z, Z(F30A) and five homologous domains (E,D,A,B, and C). The horizontal lines indicate amino acid identity. The replaced asparagines and glycine in the B domain are underlined. Z(F30A) and all its mutants include the same Nterminal as Z(F30A). Z(N23T) was constructed with the same N-terminal as Z.
A comparison of the amino acid sequence in the different homologous domains in SPA, suggests that asparagines on position 11, 23 and 43 could be replaced by serin, threonin and glutamic acid respectively. The asparagines in position 6, 21 and 28 were replaced by alanines, as no obvious alternative amino acid exists in the homologous domains. As can be seen in Figure 15, there is an asparagine in position 3. As this is in the flexible N-terminal end of the domain and outside the ordered part of the structure, it was reasoned that this would probably not contribute to the alkaline tolerance of the entire domain, and was therefore excluded from the study. However
54
this position will still be very important to investigate if multimeric constructs are desired. When the single mutants had been constructed, a study was undertaken to investigate how the introduced point mutations affected the function of the Z variants as affinity ligands. Before this investigation could proceed, a careful study was necessary to characterize how the single mutations affected structure and affinity. Structure analysis was performed using CD, and the affinity was elucidated with a Biacore where different Z domains were compared to the native Z molecule. The structural analyses using CD showed that all constructs exhibited CD spectra similar to the native Z molecule. All spectra had typical a-helical characteristics with a minimum of 208 and 222 nm, in combination with a maximum of around 195 nm. However Z(F30A, N52A) seemed to have a somewhat lower a-helical content than their parental Z molecule, and the other mutants thereof. This might be due to the postulated hydrogen bond between dO of N52 and the backbone amide proton of N21, and that the side chain of N21 is hydrogen postulated to bind the carbonyl oxygen of N52 (Starovasnik et al. 1996). Biospecific affinity interaction analysis demonstrated no dramatic effect in the affinity of any of the variants (see Table 6). As can been seen in the same table, the suppressor mutation F30A did not seem to decrease the affinity between the Z domain and IgG. Rather the opposite can be seen, which is in accordance with earlier results reporting that a small increase in affinity could be measured (Cedergren et al. 1993; Jendeberg et al. 1995).
55
Table 6. Overview of the kinetic study on the different Z domains carried out using the Biacore.
Mutant
ka
kd
KA
∆∆G ∆∆G [vs Z] [vs Z[F30A]] 5 -1 -1 -3 -1 7 -1 [10 M s ] [10 s ] [10 M ] [kcal/mol] [kcal/mol]
Z Z(N23T) Z(F30A) Z(F30A,N6A) Z(F30A,N11S) Z(F30A,N21A) Z(F30A,N23T) Z(F30A,N28A) Z(F30A,N43E) Z(F30A Z(F30A,N23T,N43E)
1.5 2.7 1.9 7.0 1.6 1.0 2.1 3.1 1.3 1.5 0.8
3.7 3.9 4.2 21 4.9 3.8 3.8 9.9 5.1 4.9 3.8
4.0 7.0 4.5 3.3 3.2 2.6 5.6 3.2 2.6 3.0 2.0
0 -0.3 -0.1 0.1 0.1 0.3 -0.2 0.1 0.3 0.2 0.4
0 0.2 0.2 0.3 -0.1 0.2 0.3 0.2 0.5
Z was used as an internal standard during the different measurements. The differences in free binding energy were calculated relative to Z and Z(F30A) respectively.
The aim of the study was to investigate the effect of the various different single mutations on the alkaline resistance of the Z domains. The results of the affinity chromatography experiments using amine coupled monomeric domains are shown in figure 16. The suppressor mutated ZF30A was still quite stable to alkaline conditions. Even after 7.5 hours of exposure to 0.5 M NaOH, the column remained functional, though with reduced capacity. The most important mutation was shown to be the asparagine to threonin substitution in the loop between helices one and two, leading to ZF30AN23T. This mutation increased the alkaline tolerance of Z(F30A) remarkably. Also, the mutation was able to stabilize the native Z molecule when it was grafted into the parental Z-domain. This agrees with earlier results showing that asparagines located in unstructured regions are more susceptible to covalent modifications than those located in structurally more inflexible regions (Kosky et al. 1999; paper IV).
56
Capacity [%] 100 90 80 Z(F30A,N23T) Z(F30A,N23T,N43E)
70 60
Z(F30A,N28T)
50
Z(F30A,N6A) Z(F30A,N11S) Z(F30A,N11S,N23T) Z(F30A,N6A;N23T) Z(F30A) Z(F30A,N43T) Z(F30A,N52A)
40 30 20 10 0
Z(F30A,N21A)
0 0
1
100
2
3
200
4
5 300
Time of Exposure
6
7 400
8
500
[h]
Figure 16. Comparison of capacity after repeated CIP-treatment with a 0.5 M NaOH cleaning agent, following an ordinary affinity chromatography scheme. The protocol was run 16 times and the duration for the alkaline exposure was 30 minutes in each round. The total time of exposure to 0.5 M NaOH was 7.5 hours.
To investigate the structural stability of Z and different mutants thereof in alkaline conditions, CD-spectra at two different pH, 7.6 and 13.7, were measured. The parental Z possesses a remarkably high stability at high pH-values, almost appearing unaffected. In addition, Z(F30A) shows lower secondary structure content in high pH solution. The results imply that the N23T-mutation slightly stabilizes the secondary structure of Z(F30A). Comparing the CD data for Z with the C2 scaffold, both domains showed high thermal stability while CD measurements in GdnHCl solutions indicated that the Z domain showed higher stability than C2 (Gülich et al. 2000). In addition, the Z domain also showed high a-helicity at high pH, while CD measurements on the C2 domain indicated no secondary structure at all in the same conditions (data not shown).
57
Concluding remarks In this thesis, attempts have been made to illustrate the benefits of using protein engineering for biotechnological applications. Discussions have emphasized sitedirected mutagenesis for exploring protein-protein interactions, and improving the performance of small protein receptor domains from bacteria. A mutagenesis strategy was applied to investigate the binding surface of the HSA binding domain originating from SPG. The residues contributing to the affinity where found to be located mainly in the second helix. Additionally, electrostatic interactions were found to be important for the association between ABD and HSA. This has also been shown to be important for other protein-protein interactions (Clackson et al, 1998; Schreiber and Fersht 1996). The localization of the HSA-binding region at ABD should provide new ways to enhance and improve ABD for different applications; including the possibility to introduce new binding specificities into the small domain, while still retaining the affinity for HSA. In the stabilization projects, three different bacterial domains were analyzed, ABD and the C2 domain originating from SPG and the Z domain originating from SPA. In all cases, the most sensitive asparagines were found to be located in loops between ahelices, or between a a-helix and a b-strand. This agrees with previous reports that the fragility of asparagine towards modifications is dependent on different secondary structure elements. In two cases, the stabilized variant of ABD, and C2 were genetically fused to provide dimers to increase the column capacity during affinity chromatography. The rational design strategies used to increase the tolerance to alkaline pH levels, clearly show upon the benefit of understanding underlying reasons for degradation. This strategy might also be applicable to increase the alkaline tolerance of other proteins and expand the use of proteinaceous ligands in different biotechnological applications.
58
Acknowledgements I would like to thank all my former and current friends and colleagues at the Department of Biotechnology; not only for all their research-related input including seminars, but also for the fantastic camaraderie we enjoyed, with “musik-kvällar”, social activities, conferences and science. I wish to specially thank the following people: Professor Mathias Uhlén, for accepting me as a PhD student, and for your inspiration over the years. Sophia Hober, my amazing supervisor. It has been a great pleasure working with you. Per-Åker Nygren for valuable discussion regarding science and for proofreading of this thesis. My laboratory colleagues at EDEN - Maria and Nina. My project support in Kaspar and Anneli, for their well performed “exjobb”, without your help I would definitely not have come this far. Thanks also to Karin, Elin and Anna at Affibody AB for valuable collaboration. To Dr Sussie Gülich for helping and guiding me through my “exjobb”. I would also like to thank all of my friends outside the lab, especially Morgan, Petra, Kaj, Lisa, Jonas, Björn J, Björn H, Annica H, Staffan, Magnus and Annica O - for lots of laughs. Also the “K93 årgång” - Stoffe, Henrik, Anna B. and Stina - imagine 11 years have gone by just like that! Thanks too to my family - Mum, Bengt, Dad, Sonja, both my brothers with families, Claes, Johan, Malin, Sunna and the juniors - Lina, Orm and Lova, for just being there. Also my “bonus” siblings with families- Petter, Anna, Eva and Mille. Good luck Anna and Mille! My thanks here also to the “Forshaga family”- Gunno, Yvonne, Martin, Markus, Zandra and Sandra. My beloved Caroline, for your love and encourage through the years. My daughter Elvira, for remanding me it is a world outside the lab. Tusen tack!
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Abbreviations ABD albumin binding domain C2 protein G domain CD circular dichroism CH2 constant heavy chain 2 CH3 constant heavy chain 3 CIP cleaning in place Cm midpoint of denaturant induced transtition C-terminal carboxy-terminal Da Dalton ELISA enzyme linked immunosorbent assay EDTA ethylenediaminetetraacetic acid Fab- fragment antigen binding Fc fragment crysttizable FRET Fluorescence resonance energy transfer Fv fragment variable GdnHCl guanidine hydrochloride HSA human serum albumin IgG immunoglobulin G ka association rate constant kd dissociation rate constant Ka affinity constant Kd dissociations constant Mab monoclonal antibodies NHS N-hydroxysuccinimide NMR nuclear magnetic resonance N-terminal amino- terminal PCR- polymerase chain reaction RU resonance unit SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SPA staphylococcal protein A SPG streptococcal protein G Tm midpoint of temperature-induced transition Z engineered protein A domain
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