A directed evolution strategy for optimized ... - Wiley Online Library

A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge MUSTAK A. KADERBHAI, HAZEL M. DAVEY,

AND

NAHEED N. KADERBHAI

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, United Kingdom (RECEIVED February 16, 2004; FINAL REVISION June 9, 2004; ACCEPTED June 9, 2004)

Abstract A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in “sequence spaces” ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of −1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli. Keywords: protein secretion; protein translocation; signal sequence; directed evolution; cytochrome b5; bioenergetics

The production of many recombinant proteins in the bacterial cytoplasm is frequently limited by their tendency to form inclusion bodies. These inclusion bodies can, however, in some cases ease the isolation of the recombinant proteins but may not yield functional renatured molecules (Fischer 1994). Correctly folded, functional recombinant proteins with a required amino terminus can be conveniently produced by means of secretion or export into the periplasmic space where there is provision of a less harsh

Reprint requests to: Mustak A. Kaderbhai, Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, UK; e-mail: [email protected]; fax: +44-1970-622294. Abbreviations: E, Energization state; m-CCCP, m-chloro carbonyl cyanide phenylhydrazone; PAGE, polyacrylamide gel electrophoresis; P, protonophoric force; SDS, sodium dodecyl sulphate; RH123, Rhodamine 123. Article and publication are at http://www.proteinscience.org/cgi/doi/ 10.1110/ps.04697304.

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environment compared with that of the cytoplasm (Wulfing and Pluckthun 1994). Protein misfolding in the periplasmic space can be countered by a slower and controlled folding rate imposed by the signal sequence and polypeptide threading through the Sec translocon. The availability in the periplasmic space of many of the essential post-translational modification enzymes catalyzing signal peptide processing (Gallagher et al. 2001), disulphide bridging (Regeimbal and Bardwell 2002), and molecular chaperones such as cytochrome c maturation factors can ensure generation of posttranslationally modified, bioactive heterologous bioproducts (Hopper et al. 2002). Moreover, in vitro permeabilization of the Escherichia coli cell wall (Kaderbhai et al. 1997) can facilitate selective discharge of the periplasmic contents into the growth medium, easing recovery of highly pure recombinant proteins. For both the useful production and study of biocatalysts and the utilization of their activity in

Protein Science (2004), 13:2458–2469. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society

Enhanced protein secretion by directed evolution

biotechnology, higher yields of authentic periplasmically localized proteins conveniently circumvent substrate/product permeability problems associated with transport across the bacterial inner membrane (Kaderbhai et al. 2001). Despite these potentials and recent insights into the mechanism of preprotein translocation (Thanassi and Hultgren 2000), the ability to overproduce secreted proteins has not kept pace with the generation of cytoplasmically localized heterologous proteins (Jonasson et al. 2002). Multiple pathways direct protein translocation across the bacterial membranes but most of the periplasmic pre-proteins are routed via the Sec- (Mori and Ito 2001) or Tat(Berks et al. 2000) export-dependent pathways. Proteins destined for translocation across the cytoplasmic membrane are synthesized as precursors carrying an amino-terminal signal sequence that direct polypeptides into the secretory pathways (Economou 1999). Although variable in primary structures (Izard and Kendall 1994), signal sequences contain a conserved and ordered structure (von Heijne 1990) that channels the passenger portion into the export pathway (Thanassi and Hultgren 2000). The amino-terminal positively charged end, together with the central hydrophobic core, directs Sec-independent and proton-motive force (PMF)-dependent signal peptide translocation across the membrane (van Voorst and De Kruijff 2000), and substitutions of the hydrophobic residues with charged ones diminish or abolish export competency of signal sequences (Silhavy et al. 1983). The efficiency of preprotein translocation per se is independent of the structure of the cleavage region. This region can accommodate varying hydrophobicities with the exception of bulky residues at −1, −3 positions (Laforet and Kendall 1991). By reducing the signal peptide to simplified, idealized segments it has been shown that a largely polymeric sequence with retention of the early consensus sequence and a central hydrophobic core, MKQST(L10)-(A6), can function equivalently to the wildtype alkaline phosphatase signal peptide (Laforet and Kendall 1991). Thus, in principle, a previously non-secreted protein can be converted into an export-competent form by appending a signal sequence at its amino terminus, but this empirical approach has not met with complete success in a biotechnological context. Comparatively few eukaryotic proteins have been reported to be efficiently hypersecreted in E. coli. Secretion parameters often derived from either pulse-chase radiolabeling and in vitro translocation assays may not necessarily be relevant to applications demanding high levels of secretion. Within a physiological context, the rate of secretion of a given polypeptide may be evolutionarily matched with structural variations in signal sequences and the early mature region of the passenger protein (Rusch et al. 1994). Indeed, there is strong emerging evidence that the amino acid composition of the early mature portion of the passenger protein beyond the signal cleavage site plays an impor-

tant role in protein translocation (Andrews et al. 1988; Li et al. 1988; MacIntyre and Henning 1990; MacIntyre et al. 1990; Struyve et al. 1993). There is a need for developing a systematic approach that can search for an ideal “sequence space” at the mature region to give dependable hyper export of a nonsecretory protein. We have developed such a simple, directed evolutionary strategy (Farinas et al. 2001) that introduces random peptide appendages between a signal sequence and the mature region of a model chromogenic hemo-protein. Positive clones secreting recombinant proteins in excess of several mg/L of culture, under standard batch growth conditions, can be conveniently traced using the PINK expression system, in turn enabling identification of ideal “algorithms” for use with a given signal sequence. We further demonstrate that discharge of pre-proteins into the periplasm by means of electrophoretic discharge across the inner membrane is principally governed by the (1) nature of charged amino acid residue(s) in a subtle blend with proximal residues in the early mature portion and (2) prevailing membrane energization state. Results and Discussion Directed evolution of secretory cytochrome b5 isoforms We used a directed evolutionary approach to generate E. coli strains expressing isoforms of secretory cytochrome b5, harboring short stretches of randomly variable amino acid sequences between a signal sequence and the mature region of the protein. The pink color of the hemoprotein facilitated visual detection of potential transformant colonies. The starting point in the construction scheme was a chimeric cytochrome b5 precursor cloned in the plasmid pAF-cyt under tight transcription control of the pho promoter. In this vector, the reading frame between the alkaline phosphatase signal sequence and the cytochrome b5 was strategically placed out of reading frame phase but can be brought into phase by inserting a segment of random oligonucleotide sequence encompassing lengths of (3n+1) nucleotides (Fig. 1). A random sequence of tetra- and heptaoligonucleotide linkers reinstates the reading frame of the secretory hemoprotein with the signal peptide, enabling search for potentially positive clone lines using the “PINK” reporter system (Kaderbhai et al. 1992). A built-in feature of the strategy is that the derivatives will encode the dipeptide Arg+1-Ile+2 at the mature portion preceding the engineered random sequences. Of the 250 randomly selected transformant colonies, 48 putative positive colonies were identified by a change in color from “buff” gray to pink. The proximal DNA regions of the inserted fragments were sequenced. Twenty-seven of these cell lines coded for different amino acid appendages. The evolved peptide sequences derived from the DNA sequencing are shown in Figure 2. Several www.proteinscience.org

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Figure 1. Construction scheme of plasmids encoding amino-terminal variant forms of cytochrome b5. BamHI–PstI-adapted synthetic oligonucleotide incorporating either a tetra or a hepta oligonucleotide of random sequence was ligated into BamHI–PstI cut pAF-CYT plasmid (Karim et al. 1993). Ppho, phoA promoter; S/D, Shine-Dalgarno sequence; SS, signal sequence.

clones gave unexpected sequences implying that these must have arisen by unusual recombination events. Characterization of the exported hemoprotein variants The growth patterns of the positive clone lines following 5 h of induction in the phosphate-limited MOPS medium were similar. Non-denaturing PAGE analyses of the periplasmic extracts of these recombinant cell lines showed pronounced appearance of novel protein bands that were absent in the control E. coli pAF cell line (Fig. 2A). In addition, the E. coli periplasmic proteins were clearly unaffected, and the chromosomally encoded, co-induced periplasmic alkaline phosphatase activities of the various strains showed no significant variation (3.5 ± 0.2 units/mg protein). The novel protein species, which show small but significant variations in their electrophoretic mobilities and qualitative amounts, migrate as the fastest-migrating components amongst the Coomassie blue-detectable periplasmic proteins. The putative identities of these bands as derivatives of cytochrome b5 were indicated by their (1) predicted properties as being relatively small, globular, compact and acidic proteins with pI ∼4.5 and (2) visibility as intense red/brown bands prior to staining. This facilitated their recovery for further characterization, identification, and determination of the first 15 amino-terminal residues by Edman degradation (Figs. 2, 3). The isolated pink proteins spectrally displayed prominent oxidized protein Soret band at 413 nm, a reduced protein Soret peak of increased absorbance at 423 nm, and visible peaks at 555 nm and 527 nm (Akhtar et al. 2003), clearly identifying them as derivatives of the evolved forms of cytochrome b5 that were correctly folded and heme-assembled with heme as holo proteins in the periplasm. Analysis of the amino acid sequences of the “evolved” appendages precisely matched with those deduced from the DNA sequences of the cloned regions in the recombinant plasmids. However, some of the isolated periplasmic cytochrome b5 isoforms revealed additional post-translocational processing of the matured proteins (Fig. 3). Although the signal sequence cleavage site precedes the immediate Arg residue, maturation occurs at the peptide bond subsequent to 2460

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this residue as reported in the previous in vivo study of some of these variant cytochrome b5 (Harding et al. 1993). The absence of arginine in the final processed product is due neither to miscleavage of the translocated precursor by the signal peptidase I nor to the nature of the early region of cytochrome b5. Rather, the selective excision of the arginine residue occurs subsequent to signal sequence deletion by an aminopeptidase-sensitive to the metal chelator, o-phenanthrolene. This aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoforms in the periplasm. However, in some cases, additional proteolysis at peptide bonds within the mature portion often yielded multiple forms of the exported products from a single construct (clones pMN-211, pMN-11, and pMN-84 in Fig. 3). Export of cytochrome b5 is determined by the nature of the evolved sequence linking signal and the mature region A comparison of the measure of the spectrally quantified hemoprotein isoforms exported to the periplasmic space with the nature of the evolved appendages (Fig. 2) identified three groups. Hyperexported hemoproteins contained an acidic residue, whereas hypoexported ones harbored a positively charged or Cys residue. Isolates expressing intermediate levels of cytochrome export contained predominantly neutral amino acid residues. However, there were some exceptions. Neutrally charged residue(s) bordering the negatively charged residue also influenced the extent of the export rates. For example, in the isolates pMN-45 and pMN10, which harbor the appendage IE(G/L)Q, the occurrence of Gly in the former (IEGQ) almost halves the export rate over that in the latter containing Leu in the same position. Introduction of a Lys residue prior to the Met+1 of cytochrome b5 in two of the hyper-exporters (pMN-144 and pMN-162) reduced export rates in the corresponding derived cell lines (pMA-144K and pMA-162K) to 70% and 80%, respectively (Fig. 3). This is in contrast to occurrence of a negatively charged residue following a positively charged amino acid in clone pMN-226. Clearly, net charge, its positioning, and the composition of the proximal se-

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Figure 2. Electrophoretic profiles of cytochrome b5 isoforms expressed in E. coli strains engineered with peptide appendages interlinking signal sequence and the amino terminus of passenger cytochrome. (A) Periplasmic proteins (䊉) resolved in non-denaturing polyacrylamide [15% (w/v)] gel; (B) the corresponding precursor pools in the isolated inner membranes separated in polyacrylamide gel (17%) containing 0.1% SDS. Vertical arrows mark protein profiles of the control E. coli pAF cell line.

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Figure 3. Display of laboratory-based evolved sequences appending an alkaline phosphatase signal sequence with cytochrome b5 and the periplasmically accumulated hemoprotein derivatives. The engineered appendages are underlined. The terminal Ala (A) residue of the alkaline phosphatase signal sequence, SS−21, and the carried-over remnant Arg (R) residue of the mature alkaline phosphatase are shown in bold. ↓SS and ↓AP denote the signal peptidase and amino peptidase processing sites, respectively. Additional processing sites are shown by ↓. Cytochrome b5 residues are italicized. The control strain pAF that expresses alkaline phosphatase signal sequence alone was used as a control. The export values, average of three determinations (SD ± 7.5), are displayed in a descending order.

quence composition appear to be important determinants. A Pro input within a neutral appendage slightly enhanced export of clone lines pMN-11 and pMN-192, compared with pMN-74. The finding that an increase in the number of positive residues at the amino terminus of the mature sequence of alkaline phosphatase severely impeded protein export in E. coli was first reported by Li et al. (1988). Similar findings have been observed with translocation of other model proteins examined by use of in vitro or by pulse-chase radiolabeling (Summers et al. 1989; Struyve et al. 1993). However, this study shows that hyper-secretory strains can be evolved by shuffling just a few residues in the early mature portion. Through introduction of a random DNA sequencing coding for just 2 and 3 amino acids, it is theoretically possible to get 400 and 16,000 variants, respectively. Of the 250 clones screened, we isolated 18% positive clones using the PINK reporter system (Kaderbhai et al. 1992), indicat2462


ing that a vast combination of the sequences can be accommodated beyond the signal sequence in the search for ideal algorithms for gaining a significant level of secretion of a recombinant protein.

Export rate of cytochrome b5 derivatives is inversely related to the membrane-bound precursor pool in transit SDS-PAGE analysis of the inner membrane proteins from the engineered strains (Fig. 2B) revealed the presence of a prominent band of about 15 kD that was identified as precytochrome b5 by its size and strong and specific immunological cross-reactivity with anti-cytochrome b5 polyclonal antibodies (data not shown). The integral membrane association of these precursors was indicated by their resistance to removal from the membranes treated with either 0.5 M


NaCl or 0.1 M Na2CO3 (Fujiki et al. 1982). Qualitatively, the electrophoretogram showed significant variations in the membrane-bound precursor pools (Fig. 2B), prompting the question as to whether this was related to the extent of the hemoprotein isoforms localized in the periplasm. Thus, an accurate measurement of the rates of precursor accumulation in the inner membranes and the corresponding secreted counterparts in the periplasmic extracts was undertaken. The complete pool of the hemoprotein in the membrane, pre-extracted with 0.1% (w/v) Nonidet P-40 and converted to holo form by exogenous heme (Gallagher et al. 1992), was accurately monitored spectroscopically. Figure 4 shows the relationship between the rates of cytochrome b5 export and the corresponding membrane-bound precursor pools in transit. The hyper-exported hemoproteins (Group A) carrying a negatively charged residue in the appendage exhibited least precursor loadings in the membranes, whereas the slowest exporters (Group C) fell in a group that included the highest amounts of membrane precursor pools. The median cluster (Group B), bearing neutrally charged residues in the evolved appendages, demonstrates an intermediate distribution of both the precursor pools and export rates. The introduction of an additional positively charged residue downstream of the negative residue in two of the fastest exporters

reduced their export rates and substantially elevated their membrane precursor pools (Group D). In a statistical survey of a diverse selection of prokaryotic exported proteins, von Heijne (1986b) reported that acidic amino acids most frequently occur around the cleavage site, especially at +2 position. E. coli alkaline phosphatase was the exception to this rule because of the presence of Lys at −2 and Arg at +1, both of which have been retained in all of the cytochrome precursors in this study; nevertheless, two additional Glu residues are present +4 and +6. Interestingly, the evolved hyper-exported cytochrome b5 appendages contain an acidic residue within a +3 to +6 window (Fig. 3), which when phased with an additional positive or replaced by a neutral or a positive charge progressively reduces export efficiency and gives rise to concomitant precursor build-ups in the membrane. Native prokaryotic signals display a net dipolar asymmetric charge distribution with net positive charge at the amino terminus and net negative charge extending into the mature portion. This dipolar structure, in combination with a central hydrophobic character and a helical conformation, is of a sufficient length to traverse the lipid bilayer (von Heijne 1986b, 1990). Following ionic interaction between the positively charged amino terminus and negatively charged phospholipids on the inner membrane side charged, looping of the signal sequence across the inner membrane can be facilitated by the membrane potential (negative inside, positive outside). Therefore, this model may explain the significantly faster rates of translocation by sequences carrying a negative charge in the early mature region. The converse argument would hold for the evolved sequences containing higher net charge in this region. However, we do not discount the roles for additional factors that are discussed below. Export rates are not limited by signal peptidase processing

Figure 4. Relationship between the rates of secretion (periplasmically localized) cytochrome b5 derivatives and the corresponding rate of accumulation of pre-cytochrome b5 pools in the inner membranes. Export rates were derived by monitoring the hemoprotein content in periplasmic extracts and isolated inner membranes from cells (20 ml induced culture) cultivated for 3, 4, and 5 h during the exponential growth phase. Groups A (crossed circles), B (inverted triangles), and C (open circles) encompass clones containing negatively charged residue(s), neutral residues, and positively charged residue(s) in the evolved appendages, respectively. Group D (crosses) denote clones, reconstructed from group A, containing an additional Lys residue prior to the first residue (Met) of cytochrome b5.

Possibilities that could account for the significant differences in the export rates displayed by the evolved peptide appendages include variations in (pre)-protein rates of synthesis, turnover, translocation, and signal sequence processing. Because the expression of all of the variant forms of cytochrome b5 was controlled through identical promoter and translation elements, the first factor seems unlikely. To test whether the half lives of the precursors or final secreted products can be influenced by the nature of the early mature region (Bachmair et al. 1986), a cocktail of protease inhibitors was included in the growth medium at set intervals during the induction regime of a selection of recombinant cell lines, under conditions that did not affect cell growth (Harding et al. 1993). This did not affect the export rates in comparison with the corresponding untreated cultures, however (data not shown). www.proteinscience.org

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Translocation and exposure of the unprocessed precursor at the periplasmic surface of the inner membrane can occur despite a block in signal peptidase activity (Kaderbhai and Kaderbhai 1996). The decreased secretion rates of the variant forms of cytochrome b5 could also arise through either weaker engagement of the precursors with the translocation channel or a decreased level of processing by signal peptidase I. To assess whether the evolved sequences had influenced the cleavability of the signal-by-signal peptidase, a selection of the precursor proteins (from Groups A–D) were isolated (Kaderbhai and Kaderbhai 1996) and their capabilities to be processed by purified E. coli signal peptidase (Kaderbhai and Kaderbhai 1996) were monitored as a function of time (Fig. 5). The precursor proteins from all of the four groupings were processed to their mature counterparts in vitro. Surprisingly, precursors bearing a net positive charge in the evolved linkages were processed more efficiently than those containing negatively or neutrally charged residues. These findings indicated that precursor processing was an unlikely limiting factor in influencing export rates. Precursors carrying positively charged residues are predominantly cytoplasmically oriented von Heijne (1986a,b) has proposed that the topology of proteins in the inner bacterial membrane is determined by a “positive-inside” rule. This rule predicts that early mature regions peppered with densely populated positive charges in a precursor would be retained on the cytoplasmic side by virtue of the prevailing membrane dipole potential. For example, amino-terminal positive residues may orient at the cytoplasmic side, whereas more distant segments carrying negative charges may be affected by the membrane electrochemical potential, allowing membrane translocation by either sec-independent or sec-dependent electrophoretic displacement (Andersson and von Heijne 1994). In this study, an increased membrane precursor pool with decreased rate of secretion correlated with net electropositivity in the early

mature region (Fig. 4), indicating that export was impeded by a mechanism similar to that postulated for the positiveinside rule (von Heijne 1986a). Thus, topologies of a selection of precursor proteins in the inverted inner membranes, representing the four groups identified in Figure 4, were assessed by the susceptibility of the globular, heme-bound domain to proteolysis. Following inactivation of trypsin by treatment with phenylmethylsulphonyl fluoride, membranes were sedimented and the trypsin-resistant globular cytochrome b5 (Strittmatter et al. 1972) was spectrally estimated in the supernatant fraction. The data presented in Figure 6 imply that poorly exported cytochrome b5 precursors are predominantly cytoplasmically disposed. Membrane energization state determines the rate of protein export It has been suggested that the membrane potential drives export of most, if not all, periplasmic proteins (Daniles et al. 1981; Samuelson et al. 2001). One model suggests that the electrochemical potential across the translocase acts as a protein/proton antiport (Duong and Wickner 1997). Is translocation of preproteins across the E. coli translocase determined electrophoretically by the nature of charged residues in the early mature region? To resolve this question, we measured the relative magnitude of membrane energization state (E) using the cationic fluorescent dye, RH123, by flow cytometric analysis (Davey and Kell 1996). Because viable bacteria accumulate the cationic lipophile in an uncouplersensitive manner, RH123 is a reliable probe for assessing the energization status of the inner membrane (Scaduto and Grotyohann 1999). The indicator dye topologically accumulates in the membrane in proportion to its energized state, generating a red shift in both absorption and fluorescence emission spectra. Using this approach, the relative RH123 distribution in the inner membrane of cells exporting various forms of cytochrome b5 was measured (Fig. 7). Insignificant fluorescent shift was noted between coupled and uncoupled states of the protonophoric force (P) in both the

Figure 5. In vitro processing of isolated precursors of cytochrome b5 isoforms by purified signal peptidase. The individual precursor proteins (50 pmoles) were incubated with E. coli signal peptidase I (3 pmole) in a final volume of 30 mL for the indicated periods and then separated by SDS-PAGE as described in the Materials and Methods section. Assays were conducted in the absence and the presence (*) of 0.4% (w/v) NP-40. The proteins were detected by staining with Coomassie blue R250.

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pendage carrying a negatively charged residue prompted further investigations, especially in view of the fact that the neutral-region-appended forms produced little or no detectable shifts either in the presence or in the absence of the uncoupler. Hence, we investigated the effect of in vivo uncoupling with mCCCP on the export of the cytochrome b5 isoforms in the four groupings (Fig. 4). For a clearer discrimination of the export rates, the analyses were conducted using E. coli cells cultured in the presence of 0.75% (w/v) glycine, which discharges periplasmically localized cytochrome b5 directly into the medium without inducing cell lysis, facilitating an easier and more reliable quantification of the hemoprotein recovered in the growth medium (Fig. 8). The inclusion of the uncoupler at increasing concentrations in the growth media at the start of culture induction caused enhanced export (approximately fivefold) of cyto-

Figure 6. Topology of pre-cytochrome b5 forms in the E. coli inner membranes assessed by limited proteolysis. Freshly isolated inverted inner membranes (250 ␮g protein), in a final volume of 100 ␮L containing 0.25 M sucrose, 10 mM Tris-HCl (pH 8.0), were treated with 0.5 ␮g tosyl phenylalanine chloromethyl ketone-treated trypsin either in the absence or the presence of 0.05% (w/v) Nonidet P-40 for 1 h at 25°C. After terminating reactions with 1 mM phenylmethylsulphonylfluoride, membranes were sedimented (200,000g for 0.5 h at 4°C) and supernatants were assayed for cleaved globular cytochrome b5 core after adding heme at 2.5 ␮M final concentration. The cytochrome b5 content at the cytoplasmic facing surface is expressed as a percentage of that derived in membranes proteolysed in the presence of the detergent at sub-lytic concentration. Bars show S.D. for three measurements.

cell lines pAF and pMN-74, expressing the signal peptide alone and cytochrome b5 with a neutral charge appendage, respectively. However, the cell line pMN-162, exporting a hemoprotein containing a negatively charged residue, displayed a significant fluorescence shift toward higher channel settings, which, however, on uncoupling with mCCCP, returned to a basal pattern seen in pAF and pMN-162. Interestingly, the cell line pMA-162K, engineered with composite positive and negative residues, diverts the fluorescence signals to opposite, lower conduits to those recorded in pMN-162, which, however, on uncoupling returned to the standard distribution seen with pMN-162. Similarly, the pMN-242 cell line expressing a cytochrome b5 form with a positive residue exhibited a pronounced, bimodal, fluorescence scatter in lower channel settings that on mCCCPmediated uncoupling again faithfully returned to the basal pattern. These findings indeed reflect the topological distributions of RH123 in the inner membrane with respect to their prevailing E states during export of the cytochrome b5 isoforms analyzed in the four groupings depicted in Figure 4. The findings that a precursor carrying a positively charged residue caused an inverse fluorescence shift of RH123 interaction compared with that observed in the ap-

Figure 7. RH123 distribution in E. coli inner membranes analyzed by flow cytometry during export of cytochrome isoforms carrying different charges at the amino terminus. Fluorescence of RH123 at 525 nm was determined in the presence (solid black line) and the absence (gray shaded line) of the uncoupler m-CCCP.


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Charge distribution at the signal-mature junction is the determinant of precursor discharge across the inner membrane

Figure 8. Effect of the uncoupling E. coli on the export of cytochrome b5 forms containing different charge appendages between the signal sequence and mature portion of the passenger protein. E. coli cells were cultivated in MOPS medium containing 0.9% (w/v) glycine (Kaderbhai et al. 1997), 5 ␮M heme, and m-CCCP at the indicated concentrations for 18 h. The medium-exported cytochrome b5 were spectrally estimated, as described in Materials and Methods, using clarified medium supernatants.

chrome isoforms carrying a positive charge (Group C) in the appendage. In the strains harboring tandemly engineered positive and negative charges (Group D), an approximately twofold increased export of cytochrome b5 was attained at 10 ␮M mCCCP. In contrast, a similar extent of decline in the rate of secretion was displayed by clones expressing cytochrome b5 isoforms carrying a negative charge in the appendage. Insignificant change in the export rates of cytochrome b5 isoforms carrying a neutral appendage was observed. Similar experiments using nigericin did not generate significant variations in the export patterns. Thus, the contribution of the ⌬pH component was negligible toward ⌬E in expectation with the rigorously maintained external pH of 7.45 with use of the MOPS-buffered growth medium. 2466


A plausible model (Fig. 9) depicting the influence of charge variations in the early mature region on the translocation of pre-protein across the inner membrane can be drawn from the findings presented here. The precursor arriving at the inner membrane surface has to gain export competency via the pre-protein translocase (PPT) with a prerequisite for transmembrane disposition of the signal sequence. We are unable to comment on the mechanism by which signal sequence disposes across the inner membrane to gain translocation competency with PPT. Nevertheless, the combined present observations unambiguously suggest an electrophoretically mediated transmembrane disposition of the signal sequence that is principally determined by the nature of charge distribution in the early sequence of the adjoining passenger protein. Optimal precursor translocation and minimal in-transit membrane loading (Fig. 4) are favored by occurrence of a negative charge proximally “dressed” with non-charged residues in the early mature portion under the prevailing P (Figs. 7, 9A) that, however, on uncoupling of E deteriorates export to a level of those bearing a neutral charge appendage (Fig. 8). The occurrence of a neutral charge at the linkage is sufficient for signal sequence to cross the membrane in a P-independent manner (Fig. 9B), albeit at a slower rate (Fig. 3) than those carrying a negative charge (Fig. 9A). Conversely, a precursor carrying a positive charge (Fig. 9C) can engage with the inner membrane/ PPT (Fig. 7), but its translocation is impeded by the unfavorable prevailing P (Fig. 7), which, on uncoupling, reinstates significant export of the processed cytochrome in the medium (Fig. 8). The extent of translocation of precursors carrying a positive charge in the appendage correlates with the pKR of the charged residue. However, this does not explain why the inclusion of Cys residue at the signal-mature junction also causes inhibition of the translocation at a level lower than that invoked by basic residues. The occurrence of a Cys residue in the early mature region of native bacterial precursor proteins is rare and suggests this constraint may be imposed by participation of a thiol moiety in the early stages of precursor-induced translocon activation. The sec-dependent polypeptide chain translocation has been shown to be dependent on membrane electrochemical potential (Dreissen and Wickner 1991). The membrane potential can specifically facilitate the initial insertion of the signal into the sec machinery, however, if negatively charged residues are present near the mature portion. Geller et al. (1993) reported that insertion of a signal sequence containing a positive charged residue in the early mature region strongly inhibited the resting membrane potential (⌿). Likewise, in this study the positively charged residues introduced at the mature region led to accumulation of the


Figure 9. Models depicting protonophore-dependent (A,C) and -independent (B) electrophoretic-like transfer of the early region of pre-cytochrome b5 across the inner membrane of E. coli. The relative positions of charged residues occurring within the alkaline phosphatase signal sequence and in the adjoining appendage linking the mature cytochrome b5 are shown. The signal sequence is shown in bold. PPT, pre-protein translocase.

unprocessed precursor with a topological orientation facing the cytoplasm (Fig. 6), which caused a redistribution of the RH123-partitioned fluorescence (Fig. 7). Interestingly, dissipation of the protein gradient (E) by the protonophore, mCCCP, reinstated the periplasmic translocation and processing of the precursors carrying positively charged residues. The precise mechanism by which uncoupling of the P reinstates the export of positively charged residue in the mature region remains to be established and a more detailed understanding of this will provide a basis for improved export of proteins. Nevertheless, the laboratory-based evolutionary approach described here yields valuable data on the features that support efficient export of the passenger protein across the inner membrane. In conclusion, these findings underpin useful applications in generating secretory proteins in a biotechnological context. Materials and methods

teria were cultured at 35°C in Luria-Bertani medium (LB) composed of 1% (w/v) Tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, containing 75 ␮g ampicillin/mL. An LB-grown saturated culture of E. coli served as a 2% (v/v) starter inoculum for induction in a phosphate-limited (0.1 mM) MOPS medium (Karim et al. 1993) at 35°C.

DNA manipulations and screening The standard operations involving digestion, ligation, analyses, and sequencing of DNA were performed as described (Ausubel et al. 2001). Plasmid DNA was isolated using the Wizard Midiprep DNA kit (Promega, Southampton, UK). Competent E. coli cells were prepared as described by Akhtar et al. (2000). PCR was performed on a Hybaid OmniGene thermal cycler using Thermus brockianus DNA polymerase. Plasmids pMA-162K and pMA144K were constructed by inverse PCR using pMN-162 and pMN144 as templates whereby a Lys residue was introduced prior to the “initiator” Met residue of the native cytochrome b5. Potentially positive transformant clones expressing cytochrome b5 isoforms were isolated using a modified MOPS PINK reporter expression system (Kaderbhai et al. 1990, 1992).

Enzymes and chemicals Subcellular fractionations Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs and Promega (UK). LabM supplied Tryptone and yeast extract. Fluka Chemicals supplied agarose and 4-morpholinesulphonic acid (MOPS). Rhodamine 123 (RH123) was purchased from Molecular Probes. Custom-synthesized oligonucleotides were supplied by MWG-Biotech AG (Germany). All other reagents were obtained from Sigma Chemicals (UK) and BDH (UK).

Bacterial culturing E. coli strains of TB-1 [F ara ⌬(lac-proAB) rps ␾80d lacZ⌬M15 thi hsdR17 rpsL (Strr)] harboring the ampicillin-resistant plasmid pAF or its derivatives were used throughout this study. The bac-

Triplicate cultures of MOPS-induced E. coli TB1 (100 mL), induced for periods stated elsewhere, were harvested by centrifugation at 5,000g for 10 min and then suspended in 20 mL of 20% (w/v) sucrose, 1 mM Na2EDTA, 0.33 M Tris-HCl at pH 8.0 (SET). Following incubation at 22°C, the re-isolated plasmolysed cells were resuspended in the residual STE volume and then osmotically shocked by dilution with 2 mL of ice-cold 0.5 mM MgCl2. Following a 10-min incubation on ice, the periplasmic fraction was recovered as the supernatant portion following centrifugation at 15,000g for 10 min at 4°C. The residual cell material was used to prepare the inverted inner membranes essentially as described (Douville et al. 1995). The crude membranes were separated by discontinuous sucrose gradient centrifugation as described (Osborn et al. 1972). The brown inner membrane band, resolved in the


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lower third part of the gradient, was diluted with an equal volume of 10 mM Tris-HCl at pH 8.0, reharvested by centrifugation at 105,000g for 1.5 h at 4°C, and finally resuspended in 2 mL of 0.25 M sucrose and 10 mM Tris-HCl at pH 8.0.

(Pharmacia-LKB, Amersham, UK) in TE buffer and eluted by applying a gradient of NaCl ranging from 0 to 0.5 M.

Electrophoretic analyses Flow cytometry

Proteins were separated by PAGE either in the absence or in the presence of SDS, using a discontinuous buffer system (Laemmli 1970) with 100 ␮g protein loading in each lane. The proteins were detected by staining with Coomassie blue R250.

A freshly prepared 1 mM solution of RH123 in ethanol was added to a 1-mL fraction of 5h MOPS-induced E. coli cells to a final concentration of 0.3 ␮M. To facilitate dye uptake, the cells were incubated at 35°C (in the dark) with gentle agitation under standard aerobic conditions for 30 min. Unstained samples were analyzed as controls. The flow cytometric analyses were performed employing a Coulter Epics Elite Flow Cytometer (BeckmanCoulter, UK) using an argon ion laser with excitation at 488 nm. The sheath fluid, composed of 150 mM KCl and 10 mM HEPES in Millipore MilliQ-filtered water, was sieved through a 0.22-␮m filter. After adjusting the pH to 6.8 with KOH, the solution was filtered through a 0.1-␮m Whatman WCN filter. Data acquisition of RH123 fluorescence was through a 525-nm band pass filter using log amplification, which yielded clear signal discrimination of the bacterial cells over the background noise.

Protein profiles were transferred from unstained polyacrylamide gels onto nitrocellulose acetate membranes (Schleicher and Schuell, Germany). Cytochrome b5 was immunologically monitored on the Western blots by sequentially probing with affinity-purified goat anti-rat cytochrome b5 IgG and alkaline phosphatase-coupled guinea pig anti-goat IgG. The immunologically cross-reactive bands were detected by the activity of alkaline phosphatase after incubation of the blot with 0.5 mg/mL ␣naphthyl pyrophosphate and 0.5 mg/mL 4-chloro-o-toluidine diazonium in 30 mM Tris-HCl at pH 9.0.

Assays

Protein sequencing

Cytochrome b5 All of the cytochrome b5 isoforms described in this study were spectrally identical to the progenitor native rat liver globular form (Gallagher et al. 1992). They were quantified from the Soret absorption peak at 423 nm in the reduced state in the presence of Na dithionite using an absorbance coefficient of 185 mM−1 ⭈ cm−1 (Akhtar et al. 2003). Absorption spectra of the bacterial subcellular fractions, appropriately diluted in 10 mM Tris-acetate (pH 8.0) buffer either in the absence or presence of 0.1% (w/v) Nonidet P-40 were monitored by scanning from 350 nm to 450 nm across a 1-cm light path cuvette. Where heme was included to convert apo cytochrome b5 to holo cytochrome b5, it was added as a stock 1 mM solution [80% (v/v) ethylene glycol, 0.1 M Tris-HCl at pH 8.2] to both the test and the reference cuvettes to give final concentrations ranging from 1 to 5 ␮M heme.

Protein Protein content of biological samples was determined using the procedure and provisions of BioRad Laboratories (Hemel Hampstead, UK), based on the published method (Bradford 1976), employing bovine serum albumin as the standard.

Alkaline phosphatase Alkaline phosphatase was assayed using the substrate p-nitrophenylphosphate (1 mM) as described previously (Karim et al. 1993).

Signal processing Signal peptidase I-catalyzed processing of isolated pre-cytochrome b5 proteins was performed as described previously (Kaderbhai and Kaderbhai 1996). The solubilized cytochrome b5 precursors, recovered by extraction of the isolated inner membranes with 20% (v/v) acetonitrile, were applied on to a FPLC Mono-Q column

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Immuno-electrophoretic analysis

Periplasmic proteins were separated in non-denaturing 14% polyacrylamide gels on a preparative scale. The color and the significantly faster mobility of the recombinant cytochrome b5 isoforms over the rest of the proteins facilitated their identification. The pink bands were dissected out and electroeluted to give holo cytochrome b5 isoforms in amounts ranging from 50 to 100 ␮g. The purity of the hemoprotein species, assessed by measure of their specific content, typically exceeded 95%. Amino terminal sequence analyses were performed on an Applied Biosystem 473A sequencer.

Acknowledgments Part of this work was supported by Senate Funds of the University of Wales Aberystwyth. We thank Drs. Kripa Aguan and Amna Karim for kindly performing the sequence analyses. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

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