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
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ABSTRACT |
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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 ( 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.
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 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 Intracellular Hydroxyproline Accumulation--
A saturated
culture of JM109 (F 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).
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 s1)
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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
1 (TGF-
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.
) 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.
) 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.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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.
View larger version (13K):
[in a new window]
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
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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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 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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The full-length human Type I 1 and
2 collagen polypeptides
(ColECol(
1) and ColECol(
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
1 gene fusion (GST-HCol), identical to GST-ColECol(
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(
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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Interestingly, neither GST-ColECol(1), GST-ColECol(
2), GST-D4,
nor GST-HCol accumulates in either hyperosmotic shock media containing
only proline or in rich media (data not shown). GST-ColECol(
1), GST-ColECol(
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-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-
1) and 32 (FN-TGF-
1). These
results, along with successful expression of GST-ColECol(
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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are:
ProRS, prolyl-tRNA
synthetase;
Hyp, trans-4-hydroxyproline;
Amp, ampicillin;
TGF-1, transforming growth factor;
GST, glutathione
S-transferase;
HPLC, high pressure liquid chromatography;
IPTG, isopropyl-1-thio-
-D-galactopyranoside..
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