Co-translational Incorporation of Trans-4-Hydroxyproline into Recombinant Proteins in Bacteria*

Douglas D. BuechterDagger, David N. Paolella§, Bridget S. Leslie, Melissa S. Brown||, Karen A. Mehos, and Elliott A. Gruskin**

From the Life Sciences Division, United States Surgical Corporation, North Haven, Connecticut 06473

Received for publication, September 12, 2002, and in revised form, October 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha 1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies.

Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitro acylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in site-specific incorporation (1) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency. In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2-4), but experimental hurdles inherent to this approach have not yet been fully overcome.

In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5) and tryptophan (6). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type synthetase. The second requirement, although it is a prerequisite for analogue incorporation, is only beginning to be addressed (4).

Escherichia coli prolyl-tRNA synthetase (ProRS)1 is particularly intriguing with respect to these considerations. ProRS will activate several proline analogues, and some of these can be incorporated into heterologously produced proteins in E. coli. These analogues include L-azetidine-2-carboxylic acid (7, 8) and 3,4-dehydroproline (9, 10). In general, proline analogues demonstrated to be incorporated in vivo into proteins are activated in vitro by ProRS at a rate approaching that of proline (11). In contrast to the proline analogues listed above, trans-4-hydroxyproline (Hyp) has been reported not to be activated by ProRS (11), and incorporation of Hyp into E. coli-produced proteins has not been demonstrated (10). In none of these cases has the intracellular concentration of the analogue necessary to support expression and incorporation into protein been determined.

Here we demonstrate that wild-type ProRS can be exploited to incorporate co-translationally trans-4-hydroxyproline efficiently in vivo into recombinant proteins. We reasoned that even a minimal rate of misactivation and misacylation of Hyp by ProRS would be sufficient for incorporation if high intracellular concentrations of Hyp could be achieved. We focused our attention, therefore, on manipulation of E. coli proline transport systems to effect the intracellular accumulation of Hyp. We show that a simple change in E. coli culture conditions results in the intracellular accumulation of Hyp to levels that support synthesis of Hyp-containing recombinant proteins. The initial goal was to produce, in E. coli, Hyp-containing human Type I collagen and collagen-like proteins. In addition to the collagens, our results should make it possible to insert Hyp into any protein synthesized in E. coli and furthermore may have applicability to other amino acid analogues.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cloning-- The gene for prolyl-tRNA synthetase was cloned from E. coli strain XL-1 Blue (Stratagene, La Jolla, CA) by polymerase chain reaction with primers designed from the published gene sequence (GenBankTM accession no. X55518). The 5' primer added a flanking NcoI recognition site and the 3' primer a flanking HindIII recognition site. The PCR product was digested with both NcoI and HindIII and ligated into NcoI/HindIII-digested pTrc99+ expression vector (Amersham Biosciences). After expression, ProRS was purified to homogeneity according to published procedures (12). The gene for the Type I alpha 1 collagen polypeptide was cloned by polymerase chain reaction of the gene from mRNA isolated from human foreskin cells (HS27/ATCC 1634) with primers designed from the published gene sequence (GenBankTM accession no. Z74615). The 5' primer added a flanking EcoRI recognition site and the 3' primer a flanking HindIII recognition site. The gene was cloned into the EcoRI/HindIII site of plasmid pBSKS+ (Stratagene), four mutations corrected using the ExSite kit (Stratagene, La Jolla, CA), the sequence confirmed by dideoxy sequencing, and finally the EcoRI/XhoI fragment was subcloned into plasmid pGEX-4T.1 (Amersham Biosciences). The genes for the collagen-binding domain of human fibronectin (13) and the mature transforming growth factor beta 1 (TGF-beta 1) (14) and mature bone morphogeneic protein (15) polypeptides were expressed from vector pTrcHis (Invitrogen). The plasmids were a generous gift from Dr. Jane Brokaw. Mannose-binding protein was expressed from plasmid pMAL-c2 (New England Biolabs, Beverly, MA). GST was expressed from plasmid pGEX-4T.1.

Amino Acid Activation Assays-- ATP-PPi exchange assays were performed at 2 mM ATP as described (16). The second order rate constant (kcat/Km) for activation of Hyp by E. coli ProRS was calculated from the slopes of plots of the initial activation rates at Hyp concentrations between 5 and 50 mM and an enzyme concentration of 5 nM. The reported value is an average of three determinations. Because radioactively labeled hydroxyproline of sufficient specific activity is not available, we could not determine the kinetics of the aminoacylation of tRNAPro with Hyp.

Bacteriology and Protein Expression-- E. coli strain JM109 was cured of its proline auxotrophy-complementing episome by treatment with acridine orange (17). Loss of the episome was confirmed by the inability of JM109 (F-) to grow on minimal medium that lacked proline. In expression experiments, cultures of JM109 (F-) that harbored the expression plasmid in Luria broth (LB) media containing 100 µg/ml ampicillin (Amp) were grown overnight. Cultures were centrifuged and the cell pellets washed twice with M9 minimal media that contained 100 µg/ml Amp and supplemented with 0.5% glucose and 100 µg/ml of all amino acids except glycine and alanine, which were at 200 µg/ml and contained no proline. The cells were finally resuspended in the above media. After incubation at 37 °C for 30 min, hydroxyproline, proline, osmolyte, or IPTG was added when appropriate. After 3-4 h, aliquots of the cultures were analyzed by SDS-PAGE.

Intracellular Hydroxyproline Accumulation-- A saturated culture of JM109 (F-) that harbored plasmid pGST-D4 in LB and contained 100 µg/ml Amp was used to inoculate 20 ml cultures of LB/Amp to A600 nm of <0.1 absorbance unit. The cultures were grown with shaking at 37 °C to A600nm between 0.7 and 1.0 absorbance units. Cells were collected by centrifugation and washed with 10 ml of M9 media. Each cell pellet was resuspended in 20 ml of M9/Amp media supplemented with 0.5% glucose and 100 µg/ml of all of the amino acids except proline. Cultures were grown at 37 °C for 30 min to deplete endogenous proline. After outgrowth, NaCl was added to the indicated concentration, Hyp was added to 40 mM and IPTG to 1.5 mM. After 3 h at 37 °C, cells from three 5-ml aliquots of each culture were collected separately on polycarbonate filters and washed twice with 5 ml of M9 media that contained 0.5% glucose and the appropriate concentration of NaCl. Cells were extracted with 1 ml of 70% ethanol by vortexing for 30 min at room temperature. Extract supernatants were taken to dryness, resuspended in 100 µl of 2.5 N NaOH, and assayed for Hyp by the method of Neuman and Logan (18). Total protein was determined with the BCA kit (Pierce) after cell lysis by three sonication/freeze-thaw cycles. The data are the means ± S.E. of three separate experiments.

Protein Chemistry and Analysis-- For purification of protein D4, crude GST-D4 was dissolved in 0.1 M HCl in a round-bottom flask with stirring. After the addition of a 2-10-fold molar excess of BrCN, the flask was evacuated and filled with nitrogen. Cleavage was allowed to proceed for 24 h, at which time the solvent was removed in vacuo. The residue was dissolved in 0.1% trifluoroacetic acid and purified on a Vydac C4 reverse phase-HPLC column (10 × 250 mm, 5 µ, 300 Å). D4 eluted as a single peak at 26% acetonitrile, 0.1% trifluoroacetic acid during a gradient of 15-40% acetonitrile, 0.1% trifluoroacetic acid during a 45-min period. Cleavage with BrCN in 70% formic acid resulted in extensive formylation of D4, presumably at the hydroxyl groups of the Hyp residues. Formylation of BrCN/formic acid-cleaved proteins has been noted previously (19). Amino acid analysis was carried out at the W. M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT) on a Beckman ion exchange instrument with post-column derivatization. N-terminal sequencing was performed at this facility on an Applied Biosystems sequencer equipped with an on-line HPLC system. Electrospray mass spectra were obtained with a VG Biotech BIO-Q quadropole analyzer by M-Scan, Inc. (West Chester, PA). Circular dichroism (CD) spectra were obtained on an Aviv model 62DS spectropolarimeter (Yale University, Molecular Biophysics and Biochemistry Department). A 1-mm path-length quartz suprasil fluorimeter cell was used. After a 10-min incubation period at 4 °C, standard wavelength spectra were recorded from 260 to 190 nm using 10-s acquisition times and 0.5-nm scan steps. For thermal melts, the temperature was raised in 0.5 °C increments from 4 to 85 °C with a 4-min equilibration between steps. Data were recorded at 221.5 nm. The thermal transition was calculated using the program ThermoDynaCD version 0.9.8, (written by P. Predki).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We reasoned that two requirements were necessary to exploit the promiscuity of E. coli ProRS to misacylate tRNAPro so that insertion of Hyp at proline codons would occur during translation. The first requirement was the ability of ProRS to activate Hyp and subsequently charge tRNAPro with Hyp. We therefore examined the activation of Hyp by ProRS and found that hydroxyproline is a substrate for activation by E. coli ProRS (Fig. 1a). The Km for Hyp is estimated to be at least 500 mM. It was not practical to assay for activation at hydroxyproline concentrations sufficiently above this concentration for a more accurate determination of the Km. The specificity constants (kcat/Km) for activation of hydroxyproline and proline by ProRS are 0.007 s-1·mM-1 and 450 s-1·mM-1 (11), respectively. The ratio of these two constants, 1.5 × 10-5, is a measure of the fitness of hydroxyproline as a substrate compared with proline (20). Thus, Hyp is activated in vitro by E. coli ProRS ~5 orders of magnitude less efficiently than proline. At 100 mM amino acid concentration, the rate of activation of alanine by ProRS is comparable with that of Hyp (Fig. 1a). The selectivity of E. coli ProRS for proline versus Hyp and alanine in the activation step is comparable with that exhibited by other E. coli aminoacyl-tRNA synthetases that activate noncognate amino acids (21). Because of experimental limitations, we were not able to measure directly the transfer of activated Hyp to tRNAPro. In several cases of misacylation, synthetase-mediated editing removes the incorrectly activated or tRNA-esterified amino acid before insertion into a protein. E. coli ProRS can edit alanine misacylated tRNAPro (22) but does not edit misacylated cysteine-tRNAPro (23). Editing has not been demonstrated for Hyp, and because E. coli ProRS is generally promiscuous and Hyp is not naturally present in E. coli, we proceeded on the assumption that E. coli ProRS would aminoacylate tRNAPro with Hyp and would not edit either Hyp adenylate or Hyp-tRNAPro.


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Fig. 1.   a, activation of Pro, Hyp, and Ala by E. coli ProRS. ATP-PPi exchange assays were performed at 2 mM ATP as described (16). The enzyme concentration was 20 nM. Amino acid concentrations are indicated. No amino acid, no amino acid control. b, intracellular Hyp accumulated by JM109 (F-) as a function of NaCl concentration in the culture media.

A second requirement must be met by in vivo approaches to amino acid analogue substitution; intracellular concentrations of the analogue sufficient to drive misacylation of the tRNA must be achieved. Because Hyp is activated poorly in vitro by ProRS compared with proline, we anticipated that high in vivo concentrations of hydroxyproline would be necessary to result in enough Hyp-charged tRNAPro to support protein synthesis. To achieve this goal, we took advantage of the phenomenon that proline and other "compatible" solutes are actively accumulated intracellularly in response to hyperosmotic shock in E. coli and other prokaryotes (24, 25). Proline porters encoded by the putP, proP, and proU genes mediate accumulation. Both proP and proU are up-regulated in response to osmotic shock (24, 26, 27). Intracellular accumulation of Hyp by E. coli has not been reported, although Hyp does partially block proline uptake by E. coli under normosmotic conditions (28). These observations led us to expect that E. coli would accumulate Hyp in lieu of proline if cultured in hyperosmotic media. Indeed, in media that lack proline but contain 40 mM Hyp, E. coli proline auxotrophic strain JM109 (F-) accumulates Hyp when cultured in increasing concentrations of NaCl (Fig. 1b). The intracellular hydroxyproline concentration is proportional to the external NaCl concentration as high as ~600 mM. At concentrations higher than 600 mM, accumulation plateaus, possibly because of saturation of the proline porters. Using a typical value for the volume of an E. coli cell (1 × 10-15 liters), the intracellular concentration of hydroxyproline at 450 mM NaCl is ~150 mM. This concentration represents an ~4-fold increase over the extracellular concentration of 40 mM and is comparable with the increases typically found for proline under osmotic shock conditions (29). Significantly, this concentration approaches the estimated lower limit for the Km of hydroxyproline activation by ProRS.

The question remained whether the intracellular hydroxyproline level in hyperosmotically shocked JM109 (F-) resulted in an adequate amount of hydroxyproline mischarged tRNAPro to support synthesis of a recombinant protein. To address this question, we tested expression of a fragment of the human Type I alpha 1 collagen chain (Fig. 2, D4) fused to the C terminus of GST in JM109 (F-) under conditions of hyperosmotic shock. The collagen fragment comprises the C-terminal 193 amino acids of the triple helical region and the 26-amino acid C-terminal nonhelical telopeptide. To preclude the possibility that expression of the highly repetitive D4 gene would be limited by differences in codon usage in E. coli compared with humans, we synthesized the D4 gene from synthetic oligonucleotides designed to reflect optimal E. coli codon usage (30, 31). Protein GST-D4 is efficiently expressed in JM109 (F-) in minimal media lacking proline but supplemented with Hyp and NaCl (Fig. 3). At a fixed NaCl concentration of 500 mM, expression is minimal at Hyp concentrations less than ~10 mM, whereas the expression level plateaus at Hyp concentrations greater than 20 mM (Fig. 3a). Likewise, at a fixed Hyp concentration of 40 mM, NaCl concentrations less than 300 mM result in little protein accumulation, and expression decreases at concentrations greater than 700-800 mM NaCl (Fig. 3b). The sodium chloride concentrations that allow for the greatest accumulation of GST-D4 roughly correspond to those that cause the intracellular concentration of hydroxyproline to increase, which suggests that expression is limited in this range by the intracellular accumulation of hydroxyproline. Either sucrose or KCl can be substituted for NaCl as the osmolyte (Fig. 3b). Thus, the osmotic shock-mediated intracellular accumulation of Hyp is the critical determinant of expression rather than the precise chemical identity of the osmolyte. Despite the large number of prolines (14 in GST and 52 in D4) in GST-D4, its size (46 kDa), and nonoptimal growth conditions, it is expressed at ~10% of the total cellular protein. Expressed proteins of less than full-length that were indicative of aborted transcription or translation or mRNA instability were not detected.


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Fig. 2.   Schematic of the amino acid sequences of ColECol(alpha 1), ColECol(alpha 2), and D4. ColECol(alpha 1) comprises the 17- amino acid N-terminal telopeptide, 338 Gly-X-Y repeating units, and the 26-amino acid C-terminal telopeptide. A unique methionine occurs at the N terminus of D4 followed by 64 Gly-X-Y repeats and the 26-amino acid telopeptide. ColECol(alpha 2) comprises the 11-amino acid N-terminal telopeptide, 338 Gly-X-Y repeating units, and the 15-amino acid telopeptide.


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Fig. 3.   Expression of Hyp-containing GST-D4 in JM109 (F-). a, dependence of expression of GST-D4 on hydroxyproline. The concentration of hydroxyproline is indicated above each lane. Osmolyte (NaCl) was added to 500 mM in each culture, and each was induced with 1.5 mM IPTG. The arrow marks the position of GST-D4. b, expression of GST-D4 in hyperosmotic media. Lanes 2 and 3 are uninduced and induced samples, respectively, each without added osmolyte. The identities and quantities of osmolyte are indicated above each of the other lanes. Hydroxyproline was added at 40 mM in each culture, and all cultures except that in lane 1 were induced with 1.5 mM IPTG. The arrow marks the position of GST-D4.

In the experiments shown in Fig. 3, we expected that Hyp would be inserted at each of the 52 proline codons of protein D4. To confirm this, GST-D4 was cleaved with BrCN at methionines within GST and at the unique methionine at the N-terminal end of D4, and D4 was purified by reverse phase HPLC. Electrospray mass spectroscopy of this protein gave a single molecular ion corresponding to a mass of 20,807 Da. This mass is within 0.05% of that expected for D4 if it contains 100% Hyp in lieu of proline. Proline was not detected in amino acid analysis of purified D4, a finding again consistent with complete substitution of Hyp for proline. To confirm further that Hyp substitution had only occurred at proline codons, we sequenced the N-terminal 13 amino acids of D4. The first 13 codons of D4 specify the protein sequence H2N-Gly-Pro-Pro-Gly-Leu- Ala-Gly-Pro-Pro-Gly-Glu-Ser-Gly. The sequence found was H2N-Gly-Hyp-Hyp-Gly-Leu-Ala-Gly-Hyp-Hyp-Gly-Glu-Ser-Gly. Taken together these results indicate that Hyp was inserted only at proline codons and that the fidelity of the E. coli translational machinery was not otherwise altered by either the high intracellular concentration of Hyp or hyperosmotic culture conditions.

One of our goals was to develop the methodology to produce Hyp-containing collagen polypeptides in E. coli. The defining feature of the collagens is the Gly-X-Y repeating tripeptide. In vertebrate fibrillar collagens, Hyp in the Y position is critical for the formation of a stable triple helical structure and subsequent fibrils (32). Trans-4-hydroxyproline is not typically found in the X position in vertebrate collagens, but is found in this position in collagens from certain invertebrate species. The peptide (Hyp-Pro-Gly)10, which contains Hyp only in the X position, does not form triple helices under conditions in which (Pro-Hyp-Gly)10 does (33). However, the influence of Hyp on triple helix stability is context-dependent, and the peptide Ac-(Gly-Hyp-Thr)10-NH2 does form a triple helix (34). Our methodology does not discriminate between proline codons and will insert Hyp at any proline codon in either the X or Y position. Given these considerations, we were interested in the structural consequences of having Hyp in both the X and Y positions as found in D4. In neutral pH phosphate buffer D4 exhibits a CD spectrum characteristic of a triple helix (Fig. 4) (35). A negative ellipticity at 198 nm and a positive ellipticity at 221 nm characterize this spectrum. When D4 was heated to 85 °C for 5 min before the CD spectrum was obtained, the magnitude of the absorbance at 198 nm was decreased and the absorbance at 221 nm was abolished (Fig. 4). This behavior is typical of the triple helical structure of collagen (35). A thermal melt profile of D4 in phosphate buffer showed a melting temperature of 29 °C. A fragment of the C-terminal region of the bovine Type I alpha 1 collagen chain comparable in length to D4 forms homotrimeric helices with a melting temperature of 27 °C (36). Thus, despite global Hyp for proline substitution in both the X and Y positions, D4 forms triple helices of stability similar to comparably sized fragments of bovine collagen containing Hyp at the normal percentage and only in the Y position. These results support the notion that Hyp in the X position does not significantly destabilize the triple helix a priori but may, in specific situations, contribute to triple helix stability. Because Ac-(Gly-Hyp-Thr)10-NH2 forms a triple helix but (Hyp-Pro-Gly)10 does not, Pro in the Y position may counteract the otherwise stabilizing effect of Hyp in the X position (34). D4 contains Gly-X-Y triplets that place Hyp in multiple contexts with respect to neighboring amino acids; however, because of global Hyp for Pro substitution no triplets occur with contiguous Hyp and Pro. This fact, along with the overall increase in Hyp content in D4, may also contribute to its triple helix stability.


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Fig. 4.   CD spectra of native and heat-denatured D4 in neutral phosphate buffer.

The full-length human Type I alpha 1 and alpha 2 collagen polypeptides (ColECol(alpha 1) and ColECol(alpha 2), Fig. 2), although more than four times the size of D4, also express as N-terminal fusions with GST in JM109 (F-) in Hyp/NaCl media (Fig. 5a). Like D4, the genes for the collagen portions of these proteins were constructed from synthetic oligonucleotides designed to mimic codon usage in highly expressed E. coli genes. In contrast, expression from a GST-human Type I alpha 1 gene fusion (GST-HCol), identical to GST-ColECol(alpha 1) in coded amino acid sequence but containing the human codon distribution, could not be detected in Coomassie Blue-stained SDS-PAGE of total cell lysates of induced JM109 (F-)/pHCol cultures (Fig. 5a). Thus, sequence or structural differences between the genes for ColECol(alpha 1) and HCol are critical determinants of expression efficiency in E. coli. This fact is likely the result of codon distribution in these genes and ultimately of differences in tRNA isoacceptor levels in E. coli compared with humans. However, additional effects on other transcription or translation steps cannot be excluded definitively.


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Fig. 5.   Incorporation of hydroxyproline into proteins expressed in E. coli. a, expression of GST-HCol, GST-ColECol(alpha 1), and GST-ColECol(alpha 2) in JM109 (F-). Hyp was added at 40 mM and NaCl at 500 mM. Expression was induced with 1.5 mM IPTG. The arrows mark the positions of GST-ColECol(alpha 1) and GST-ColECol(alpha 2). b, expression of Hyp-containing proteins in JM109 (F-). The protein and added media components are indicated above each lane. The migratory position of each protein is indicated. The asterisk indicates an unidentified E. coli protein that is up-regulated under these culture conditions.

Interestingly, neither GST-ColECol(alpha 1), GST-ColECol(alpha 2), GST-D4, nor GST-HCol accumulates in either hyperosmotic shock media containing only proline or in rich media (data not shown). GST-ColECol(alpha 1), GST-ColECol(alpha 2), and GST-D4 are insoluble when expressed in Hyp-containing media at either 37 or 30 °C, and insolubility may segregate them from degradation pathways. Alternatively, the Hyp-containing proteins may escape degradation because they adopt a protease-resistant structure, whereas the proline-containing proteins do not. The large number of codons nonoptimal for E. coli found in the human gene and the instability of proline-containing collagen polypeptides in E. coli may, in part, explain why expression of human collagen in E. coli has not been reported previously.

We sought to generalize our observations further by determining the expression of other proteins in Hyp/NaCl-containing media (Fig. 5b). In addition to GST, mannose-binding protein, the mature TGF-1 polypeptide, a 70-kDa fragment of human fibronectin, a chimera of fibronectin and the mature TGF-beta 1 polypeptide, and a chimera of fibronectin and the mature bone morphogeneic protein 2 polypeptide are expressed in JM109 (F-) in Hyp/NaCl. In each case, expression is dependent on NaCl and Hyp. The number of prolines in these proteins varies between 9 (TGF-beta 1) and 32 (FN-TGF-beta 1). These results, along with successful expression of GST-ColECol(alpha 1) containing 255 prolines, suggest that any number of prolines can be substituted with hydroxyproline, provided the protein accumulates in E. coli. Incorporation does not depend on the post-translational enzymatic oxidation of proline to hydroxyproline, and as expected for a mechanism operating at a translational rather than post-translational level, successful substitution does not depend on the precise sequence, structure, or activity of the recombinant protein.

In media that contain a mixture of proline and hydroxyproline, the choice of which of these two amino acids is inserted at a given proline codon during translation should depend on the relative efficiency of ProRS-catalyzed aminoacylation of tRNAPro with the amino acid. Thus, selection of the appropriate ratio of proline to hydroxyproline in the growth media should make it possible to express proteins that contain any desired amount of hydroxyproline. To determine whether this is the case, we expressed GST in JM109 (F-) in media containing a fixed amount of hydroxyproline (40 mM) but increasing amounts of proline (Fig. 6). In medium containing either no proline or 1 µM proline, the migration positions of GST are identical, suggesting that these proteins contain comparable amounts of hydroxyproline. At 2.5 mM proline, GST migrates at the same position as GST expressed in media containing proline but no Hyp. At intermediate proline concentrations in the range of 1 to 30 µM, the migratory position of the protein is between these two extremes, which suggests that these proteins contain variable amounts of Hyp and proline. We estimate that 50% substitution of hydroxyproline for proline occurs at 10 µM or less proline in media containing 40 mM Hyp. The ratio of proline to hydroxyproline in the extracellular media at this concentration is 2.5 × 10-4. This ratio is close to the ratio of the in vitro specificity constants for activation of these two amino acids by ProRS (see above), which suggests that, in the absence of editing, the incorporation selectivity reflects the in vivo kinetics of activation and/or aminoacylation. Precise control of the degree of hydroxyproline substitution, coupled with genetic approaches to insert site-specific Hyp at the Y-position of the Gly-X-Y repeat, may make it possible to produce human collagens with any desired Hyp composition.


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Fig. 6.   Expression of GST containing varying amounts of Hyp and proline. All cultures, except the no amino acid control culture, contained 40 mM Hyp and the indicated concentrations of proline. Neither Hyp nor proline were added to the control. All cultures were induced with 1.5 mM IPTG.

This system is an experimentally simple and robust way to insert Hyp into any proline-containing protein that can be synthesized in E. coli. The power of the method results from the use of DNA coding triplets and the wild-type amino acid synthetase/tRNA pair. In the general case, it is likely that only modest misacylation of a tRNA with an analogue is required, provided a sufficient intracellular concentration of the amino acid analogue can be achieved. Manipulation of amino acid transport systems to achieve intracellular accumulation of the analogue may be of general use and may be feasible with amino acid analogues other than Hyp. Other organisms, including Saccharomyces cerevisiae (37), alter metabolism in response to environmental stresses, and conditions may be found in these systems to promote the cellular uptake, accumulation, and incorporation of novel amino acid analogues.

Strategically placed prolines can increase protein thermostability, and isomerization of proline residues can be the rate-limiting step in protein folding (38, 39). Evidence (40, 41) suggests that inductive effects of electronegative substituents on the proline ring affect the rate and equilibrium position of cis-trans isomerization and, consequently, triple helix stability. These effects are not likely to be specific to collagen and suggest that hydroxyproline may enhance stability or affect the rate of protein folding in noncollagenous proteins. The ability to produce large quantities of Hyp-containing proteins in E. coli should make possible biophysical and biochemical approaches to these areas and may open the door to novel proteins with unique therapeutic, biomaterial, or bioengineering applications.

    ACKNOWLEDGEMENTS

We thank R. S. Lloyd (University of Texas), K. Musier-Forsyth (University of Minnesota), J. O. Hollinger (Oregon Health Sciences University), P. Schimmel (Scripps Research Institute), N. Tawil, D. Gies, and T. Turecek for thoughtful discussions and comments on the manuscript. We also thank Michael R. Russo for assistance with the figures.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Achillion Pharmaceuticals, New Haven, CT 06511.

§ Present address: Pfizer Corporation, Groton, CT 06340.

|| Present address: Alexion Pharmaceuticals, Cheshire, CT 06410.

Present address: Bayer Corporation, West Haven, CT 06516.

** To whom correspondence should be addressed: Acorda Therapeutics, Inc., 15 Skyline Drive, Hawthorne, NY 10532. Tel.: 914-347-4300, ext. 139; E-mail: egruskin@acorda.com.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M209364200

    ABBREVIATIONS

The abbreviations used are: ProRS, prolyl-tRNA synthetase; Hyp, trans-4-hydroxyproline; Amp, ampicillin; TGF-beta 1, transforming growth factor; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; IPTG, isopropyl-1-thio-beta -D-galactopyranoside..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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