Recombinant expression systems for the production ...

Biochemical Society Transactions (2000) Volume 28, part 4

Recombinant expression systems for the production of collagen N. J. Bulleid1, D. C. A. John and K. E. Kadler School of Biological Sciences, Wellcome Trust Centre For Cell-Matrix Research, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K. Gly-X-Y triplet, where X and Y can be any amino acid but are frequently the imino acids proline and hydroxyproline. An important feature of fibrilforming collagens is that they are synthesized as precursor procollagens containing globular N- and C-terminal extension propeptides. The biosynthesis of procollagen is a complex process involving a number of different post-translational

Abstract The ability of triple-helical collagen molecules to assemble into supramolecular structures forms the basis of commercial uses of collagen in the food industry and in medical applications such as cosmetic surgery and tissue repair. We have used cDNA techniques to engineer novel collagens with potentially enhanced biological properties ; however, expression of fully functional novel molecules is difficult due to the complex nature of procollagen biosynthesis. This article outlines the application of various expression systems to procollagen production and details the use of the mammary gland as a suitable bioreactor for the synthesis of significant amounts of novel procollagens from cDNA constructs.

Figure 1 Initial stages in the folding and assembly of procollagen Three distinct stages can be defined in the folding and assembly pathway of procollagen. The first stage is the folding of the Cpropeptide, giving rise to a globular domain. Three C-propeptides can then associate during the second stage to form trimers. This association allows the third stage to occur, i.e. nucleation of the Gly-X-Y repeat region and folding of the triple-helical domain. The triple-helical structure is stabilized by post-translational modification of proline residues to hydroxyproline.

Introduction The ability of proteins to self assemble into macromolecules is fundamental to all living organisms. Some of the largest and most complex of these structures are the fibrils formed from the triple-helical protein collagen [1]. The fibrils are the main constituents of the extracellular matrix that underpins tissue assembly. Collagen and gelatine (denatured collagen) are used commercially to form gelling agents in the food industry, bulking agents in cosmetics and in corrective surgery, and to assemble artificial matrices to support cell growth during tissue repair. These industrial and medical uses are restricted to the type and availability of collagen molecules found naturally. We have developed the technology to construct collagen molecules with novel chain compositions and domain modifications that potentially will have enhanced biological properties. The only way of producing such engineered collagen molecules will be by recombinant technology.

Synthesis of collagen All fibrillar collagens molecules contain three polypeptide chains constructed from a repeating Key words : procollagen, recombinant protein, tissue engineering, transgenic animals. Abbreviation used : P4H, prolyl 4-hydroxylase. 1 To whom correspondence should be addressed (e-mail neil.bulleid!man.ac.uk).

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modifications including proline and lysine hydroxylation, N-linked and O-linked glycosylation and both intra- and inter-chain disulphide-bond formation. The enzymes carrying out these modifications act in a co-ordinated fashion to ensure the folding and assembly of a correctly aligned and thermally stable triple-helical molecule [2]. Procollagen folds and assembles through a series of distinct intermediates that can be characterized by their extent of modification, disulphide-bonding status and polymeric state (Figure 1). As the polypeptide chain is co-translationally translocated across the membrane of the endoplasmic reticulum, hydroxylation of proline and lysine residues occurs within the Gly-X-Y repeat region [3]. Once the polypeptide chain is fully translocated into the lumen of the endoplasmic reticulum the C-propeptide folds [4]. Three proα chains then associate via their C-propeptides to form a trimeric molecule allowing the Gly-X-Y repeat region to form a nucleation point at its Cterminal end, ensuring correct alignment of the chains. The Gly-X-Y region then folds in a C-toN direction to form a triple helix. The temporal relationship between polypeptide chain modification and triple-helix formation is crucial as hydroxylation of proline residues is required to ensure stability of the triple helix at body temperature [5], and once formed the triple helix no longer serves as a substrate for the hydroxylation enzyme [6]. The C-propeptides (and to a lesser extent the N-propeptides) keep the procollagen soluble during its passage through the cell. Removal of the propeptides by procollagen N- and C-proteinases lowers the solubility of procollagen by 10 000-fold and is necessary and sufficient to initiate the self-assembly of collagen into fibres [7]. Crucial to this assembly process are short nontriple-helical peptides called telopeptides at the ends of the triple-helical domain, which ensure correct registration of the collagen molecules within the fibril structure and lower the critical concentration for self-assembly [8].

reviewed in [3]) is critical for triple-helix stability. Hence, traditional high-yield expression systems such as bacteria and yeast that do not contain any P4H activity have not been used for collagen production without modification of the host. By co-expressing recombinant type-III procollagen together with mammalian P4H in the yeast Pichia pastoris, it has recently been demonstrated that triple-helical procollagen (the soluble precursor of collagen containing N- and C-propeptides) can be produced [9]. Similarly, although insect cells possess a very low level of endogenous P4H activity, co-transfection of such cells with a combination of baculovirus vectors encoding human type-III procollagen and both α- and β-subunits of P4H yielded up to 60 µg\ml cellular procollagen [10]. However, when expressed in yeast or insect cells, the protein is not secreted and must be extracted from the cells by treatment with pepsin, a protease that does not digest the triple-helical domain but does damage the telopeptides. As a consequence, pepsin-extracted collagen is unable to form ordered fibrillar structures [8]. Mammalian cell lines that possess adequate levels of endogenous P4H have also been used to express procollagens. Yields vary between the type of cell line used and the expression vector and range from relatively low levels using the HT1080 cell line (0.35–2 µg\ml) to higher levels (15 µg\ml) using the HEK293 cell line [11–13]. The advantage of this approach is that the procollagen is secreted into the medium from where it can be purified. The HT1080 cell line has only low levels of proteinase activity in the medium so most of the secreted material is procollagen, whereas HEK293 cells do contain a protease activity that cleaves the C-propeptide [14]. The material secreted from mammalian cell lines can be purified and used to form ordered fibrils in vitro by the addition of the C- and N-proteinases [15]. A summary of the various expression systems and their advantages and disadvantages is presented in Table 1. One of the most promising mammalian expression systems to date is that which utilizes the mammary gland as a bioreactor [16]. Human proteins destined for clinical use have been produced in biologically active form by expression in the milk of transgenic animals. This system utilizes either genomic inserts or cDNA transgene sequences in conjunction with mammary glandspecific promoters to drive expression of proteins in milk. The use of genomic constructs generally results in relatively high expression levels ( 1 mg\ml) and yields of up to 33 mg\ml have

Recombinant expression systems Synthesis of recombinant collagen presents various problems, not least because of the many posttranslational modifications (some of which are unique to collagens) required for an expressed collagen molecule to achieve a fully folded, triplehelical conformation. Eight specific enzymic activities are required [1] and of these the modification carried out by prolyl 4-hydroxylase (P4H ;

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Biochemical Society Transactions (2000) Volume 28, part 4

Table 1 Comparison of the various recombinant expression systems for the production of collagen Expression host

Protein expressed

Yield (µg/ml)

Advantages

Yeast (Pichia pastoris)

proα1(III) + α- and β-subunits of P4H

15

High yield, inexpensive

Insect cells

proα1(III) + α- and β-subunits of P4H proα1(II), proα1(I), proα1(III)

60

High yield

0.35–2

HEK 293-EBNA

proα1(V)

15

Transgenic animals

Modified procollagens + αand β-subunits of P4H

150

Secreted, authentic product, no need for co-expression of P4H High yields, secreted, authentic product, no need for co-expression of P4H High yield, authentic product

HT1080

been documented [17]. In contrast, expression levels from cDNAs are generally lower than those from comparable genomic sequences ( 1 mg\ml, e.g. [18]). In order to test the mammary gland expression system for the synthesis of procollagens we generated several mouse lines expressing a recombinant chimaeric procollagen molecule, corresponding to an engineered proα2(I) chain. Mouse lines were generated by integration of cDNA coding for this molecule into the mouse genome using a vector designed to express cDNA constructs in milk. Transgenic lines were established containing cDNAs encoding the α- and β-subunits of P4H together with the truncated procollagen. We have shown that up to 200 µg\ml of recombinant procollagen was produced in mouse milk and that the protein was expressed as disulphidelinked trimers containing a thermostable triplehelical domain [19].

Not secreted, low hydroxylysine content Not secreted Low yields

Some cleavage of propeptides

High development costs

in the production of correctly folded collagen that can be used as a substitute for collagen extracted from tissues. Novel engineered collagens have also been produced using either mammalian cell-lines or transgenic animals. In the future these recombinant proteins will be used both to investigate the molecular basis and biochemistry of collagen assembly and to produce collagens with new pharmaceutical and medical uses. This work was funded by grants from the Wellcome Trust, the MRC and The Royal Society. The transgenic animal work was carried out in collaboration with PPL-therapeutics Ltd.

References 1 Kadler, K. (1995) in Extracellular Matrix 1 : Fibril-Forming Collagens. Protein Profile 2 (Sheterline, P., ed.), pp. 491–619, Academic Press, Oxford 2 Prockop, D. J. and Kivirikko, K. I. (1995) Annu. Rev. Biochem. 64, 403–434 3 Kivirikko, K. I., Myllyla$ and Pihlajaniemi, T. (1992) in Posttranslational Modification of Proteins, (Harding, J. J. and Crabbe, M. J. C., eds), pp. 1–51, CRC Press, Boca Raton 4 Doege, K. J. and Fessler, J. H. (1986) J. Biol. Chem. 261, 8924–8935 5 Berg, R. A. and Prockop, D. J. (1973) Biochem. Biophys. Res. Commun. 52, 115–120 6 Berg, R. A. and Prockop, D. J. (1973) Biochemistry 12, 3395–3401 7 Kadler, K. E., Hojima, Y. and Prockop, D. J. (1987) J. Biol. Chem. 262, 15696–15701 8 Leibovich, S. J. and Weiss, J. B. (1970) Biochim. Biophys. Acta 214, 445–454

Summary The complex nature of the biosynthesis of collagen makes it a challenging protein to express in recombinant form. The requirement for the posttranslational modification of proline to form hydroxyproline is the major consideration when choosing a host to produce collagen. Co-expression of P4H in host cells lacking this enzyme has resulted


Disadvantages

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16 Colman, A. (1998) in Biochemical Society Symposium, vol. 63 (Rudland, P. S., Fernig, D. G., Leinster, S. and Lunt, G. G., eds), pp. 141–147, Portland Press, London 17 Carver, A. S., Dalrymple, M. A., Wright, G., Cottom, D. S., Reeves, D. B., Gibson, Y. H., Keenan, J. L., Barrass, J. D., Scott, A. R., Colman, A. and Garner, I. (1993) Biotechnology 11, 1263–1270 18 Prunkard, D., Cottingham, I., Garner, I., Bruce, S., Dalrymple, M., Lasser, G., Bishop, P. and Foster, D. (1996) Nat. Biotechnol. 14, 867–871 19 John, D. C. A., Watson, R., Kind, A. J., Scott, A. R., Kadler, K. E. and Bulleid, N. J. (1999) Nat. Biotechnol. 17, 385–389

9 Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T. and Kivirikko, K. I. (1997) EMBO J. 16, 6702–6712 10 Lamberg, A., Helaakoski, T., Myllyharju, J., Peltonen, S., Notbohm, H., Pihlajaniemi, T. and Kivirikko, K. I. (1996) J. Biol. Chem. 271, 11988–11995 11 Fertala, A., Sieron, A. L., Ganguly, A., Li, S.-W., Ala-Kokko, L., Anumula, K. R. and Prockop, D. J. (1994) Biochem. J. 298, 31–37 12 Geddis, A. E. and Prockop, D. J. (1993) Matrix 13, 399–405 13 Fichard, A., Tillet, E., Delacoux, F., Garrone, R. and Ruggiero, F. (1997) J. Biol. Chem. 272, 30083–30087 14 Imamura, Y., Steiglitz, B. M. and Greenspan, D. S. (1998) J. Biol. Chem. 273, 27511–27517 15 Fertala, A., Holmes, D. F., Kadler, K. E., Sieron, A. L. and Prockop, D. J. (1996) J. Biol. Chem. 271, 14864–14869

Received 28 February 2000

Expression of recombinant human type I–III collagens in the yeast Pichia pastoris J. Myllyharju1, M. Nokelainen, A. Vuorela and K. I. Kivirikko Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, P. O. Box 5000, FIN-90014 Oulu, Finland

Abstract

Introduction

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the α- and β-subunits of prolyl 4hydroxylase. The triple-helical type-I, -II and -III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g\l depending on the collagen type.

The collagen family consists of about 20 proteins formally defined as collagens and more than 10 additional proteins with collagen-like domains [1–3]. Type-I collagen is now used as a biomaterial in numerous medical applications and as a delivery system for various drugs [4–6]. In addition, all gelatins are made from collagens. The collagens used in all these applications have been isolated from animal tissues and are liable to cause allergic reactions in some subjects and carry a risk of disease-causing contaminants. The various collagen types have different properties, and therefore some of the other collagens might be more suitable for certain applications than type I. However, it has been difficult or impossible to isolate sufficient quantities of the other collagens from animal tissues. It is obvious, therefore, that an efficient large-scale recombinant system for the production of human collagens would have numerous applications in medicine. Most recombinant systems now available for large-scale production of proteins cannot be used as such for the production of recombinant collagens, as bacteria and yeast have no prolyl 4hydroxylase activity, and insect cells [7] and the mammary gland [8] have insufficient levels of this enzyme activity. Prolyl 4-hydroxylase, an α β # # tetramer in vertebrates, plays a central role in the synthesis of all collagens, as the 4-hydroxyproline residues formed are essential for the folding of the newly synthesized collagen polypeptide chains into triple-helical collagen molecules [2,9,10].

Key words : methylotrophic yeast, procollagen, prolyl 4-hydroxylase. Abbreviations used : αMF, α mating factor ; proα1(I), proα1(II) and proα1(III) chains, proα1 chains of type-I, -II and -III procollagen, respectively ; proα2(I) chain, proα2 chain of type-I procollagen. 1 To whom correspondence should be addressed (e-mail johanna.myllyharju!oulu.fi).

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