LI Su-Xia et al.: Expression of C-peptide Multiple Gene Copies in Escherichia coli

　

LI Su-Xia^1,2,
TIAN Li-Ping², LIU Hai-Feng¹, ZHANG Yu-Jian²,
HU Xiao-Bo², GONG Yi^2*，
YUAN Qin-Sheng^1*

(¹State Key Laboratory
of Bioreactor Engineering, East China University of Science and Technology,
Shanghai 200237, China; ²Research Center of Biotechnology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200233, China)

Abstract    A gene fragment encoding three copies of proinsulin
C-peptide was synthesized and expressed in E. coli and the recombinant
proinsulin C-peptide was produced through site-specific cleavage of the resulting
gene products. The fusion protein was expressed at high level, about 80 mg/L,
as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently
separated the expressed fusion protein from the supernatant, to obtain about
37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin
and carboxypeptidase B of the fusion protein efficiently released native C-peptide,
the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L.
The good agreement of amino acids composition, together with shown similarities
of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC
analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant
C-peptide obtained in this report was the native human C-peptide. The investigation
of the chemical stability of recombinant human C-peptide in aqueous solutions
by RP-HPLC was also reported. The degradation of the recombinant C-peptide
showed a marked dependence on pH and temperature. The degradation reaction
of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation
reaction of C-peptide followed first-order kinetics in pH 3 buffered solution
at 37 ℃ or 70 ℃, only 40.3% of C-peptide was remained after 10 h at 70 ℃.
The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were
detected at pH 7.4 and 37 ℃ after 10 h and at pH 7.4 and 70 ℃ after 5 h. 99%
and 96% of C-peptide was remained at pH 7.4 and 37 ℃ after 10 h with and without
10 g/L BSA respectively.



Key words      

proinsulin C-peptide; multiple
copies; expression; stability; RP-HPLC

The
proinsulin C-peptide had long been considered a by-product of insulin biosynthesis,
and it was important in the folding of proinsulin by providing a spacer sequence
that could be removed once folding was completed［1，2］. After the discovery
of the model of insulin biosynthesis, several early studies addressed the
question of possible physiological effects of C-peptide, for example, insulin-like
effects on blood glucose levels and on glucose disposal after glucose loading
were looked for but not found［3，4］. However, recent reports showed that it
elicited both molecular and physiological effect, suggesting that it were
a hormonally active peptide. Special binding of C-peptide to the plasma membranes
of intact cells and to detergent-solubilized cells had been shown, which indicated
the existence of a cell surface receptor for C-peptide［5］. Data now indicated
that C-peptide in the nanomolar concentration range bound specifically to
cell surfaces, probably to a G protein-coupled membrane receptors, with subsequent
activation of Ca2+-dependent intracellular signaling pathways［6，7］. The binding
was stereospecific, and no cross-reaction was seen with insulin, proinsulin,
insulin growth factors I and II, or neuropeptide Y［8］. C-peptide elicited
a number of cellular responses, including Ca2+ influx［9］, activation of mitogen-activated
protein (MAP) kinase[10], activation of Na+, K+-ATPase[11] and endothelial
NO synthase［12］. Data also indicated that C-peptide administration was accompanied
by augmented blood flow in skeletal muscle and skin［13］, diminished glomerular
hyperfiltration, reduced urinary albumin excretion［14]］, and improved nerve
function［15]］, in all patients with type 1 diabetes who lacked C-peptide,
but not in healthy subjects. The possibility existed that C-peptide replacement,
together with insulin administration, might prevent the development or retard
the progression of long-term complication in C-peptide-deficient type 1 diabetes
patients［16］.Production strategies for recombinant peptides constituting small
portions of larger fusion proteins generally gave relatively low yields of
the target peptide. In the presented process, C-peptide constituted only 19%
of the fusion protein. In order to increase the amount of produced target
peptide, one strategy was to synthesize a gene product with multiple copies
of the target peptide［17］. Multimerization of gene fragments could be achieved
by a number of methods. A technique for the polymerization and assembly of
peptides involved the use of the class-IIs restriction enzyme BspMI,
which enabled unidirectional insertion of the DNA fragments to be polymerizated［18］.
Two identical class-IIs restriction enzyme sites were inversely oriented in
each of two different amplification vectors with the same cut sites, complementary
cohesive ends were created to make high-copy-number multimers of DNA fragments
in a tandem unite and up to 108 copies were constructed［19］. Head-to-tail
polymerizations of synthetic DNA fragments had been used in several reports
to obtain gene multimerization by design of single-stranded non-palindromic
ends. The DNA fragments were polymerized and could then be directly ligated
to matching protrusions resulted from restriction enzyme digestion［20］. Alternative
strategies involved polymerization of the gene construct and ligation of adapter
molecules containing restriction sites to allow further subcloning［21］. When
synthesizing gene products containing multiple copies of a target peptide,
the yield of the fusion protein with target peptides did not necessarily need
to decrease. Therefore, the molar yield of the target peptide could be significantly
increased. However, in order to recover the free native peptide, the synthesized
gene product had to be processed, by chemical cleavage methods or by proteolytic
enzymes, to release the native peptide.In this paper, partly based on a strategy
of Jonasson et al.［22］, multiple copies of proinsulin C-peptide was
obtained through introducing the restricted enzymatic site, SfiI, to
ensure the multiple C-peptide-encoding gene ligated head-to-tail. Expression
vector pET-32a carrying one or three C-peptide-encoding gene fragments were
constructed and were expressed at high level in E. coli as a fusion
protein, C-peptide was recovered by trypsin+carboxypeptidase B enzymatically
digestion and further purification with RP-HPLC.
There was few fundamental information on the chemical stability of C-peptide.
This note described the pattern of degradation of C-peptide as a function
of pH and temperature by RP-HPLC.

1 Materials and Methods
1.1 Materials
All primers were synthesized by Bioasia Co. Ltd.; restriction enzyme SfiI
was obtained from NewEngland Biolabs; general vector pMD18-T, restricted enzymes
EcoRI and HindIII, ligation kit and CIAP were purchased from
TaKaRa (Dalian) Co. Ltd.; IMMULITE C-Peptide kit was product of Diagnostic
Products Corporation in USA; recombinant carboxypeptidase B was produced in
our laboratory; other plasmids and strains were stored in our laboratory.
1. 2 DNA constructions
Partly based on a strategy of Jonasson et al.［22］, briefly, two single
strands DNA, partly complementary to each other (indicated italic), were synthesized
according to the bias for preferred codons of E. coli, C-p s (84 bp):
5′-CCG GAA TTC CAG GCC TCC CAG GCC CGC GAA GCT GAG GAC CTG CAG GTT
GGT CAG GTT GAA CTG GGC GGT GGC CCG GGT GCA GGC– 3′ ( containing EcoRI
and SfiI restricted enzyme sites at 5′ end and arginine-encoding gene
indicated with bold); C-p a (77 bp) : 3′- C CCA CGT CCG AGA
AAC GTC GGC GAC CGA AAC CTT CCA AGA GAA GTC GCA TGC CGG AGG GTC CGG
TAA TTT CGA AGC G-5′ (containing SfiI and HindIII restricted
enzyme sites at 5′ end and arginine-encoding gene indicated with bold). Two
ssDNA gene fragments ( per 20 μg) was allowed to extend and anneal in the
presence of 1 u Pyrobest polymerase (TaKaRa) and dNTP as following: 30 ℃ for
5 min, then 72 ℃ for 5 min, 4 cycles; then incubated at 72 ℃ for 20 min. The
recovered double strand DNA was ligated to the general vector pMD18-T, the
ligation mixture was transformed to the dcm-E. coli strain JM110 to
allow efficient SfiI digestion. The purified C-peptide-encoding gene
fragment was allowed to polymerized in a head-to-tail fashion, and were then
ligated to the purified SfiI digested plasmid which had been dephosphorylated
with CIAP (calf intestinal alkaline phosphatase) to prevent self-ligation.
E. coli JM105 cells were transformed with the ligation mixture, and
the resulting transformants were screened using a PCR-screening technique.
Gene fragments encoding one, two and three concatamerized C-peptides were
isolated and ligated to expression vector pET-32a through EcoRI and
HindIII enzymatic sites, and the resulting plasmids were denoted pET-32a-cp1,
pET-32a-cp2 and pET-32a-cp3, respectively.
1. 3 Production and affinity purification of the fusion protein
The expression vectors were transformed into E. coli HMS174(DE3), the
cells harboring the desired expression vectors were grown individually for
a pre-culture at 37 ℃ overnight in LB medium containing 100 μg/L ampicillin.
The culture was inoculated into LB medium containing 100 μg/L ampicillin,
the cells were grown until A600 reached 0.3－0.5,
then 1 mol/L IPTG was added, after 3 h, the cells were harvested by centrifugation
at 4 ℃, 10 000 r/min for 15 min. The collected cells were resuspended in 50
mL NTA-20 (20 mmol/L Tris・HCl pH 7.9, 0.5 mol/L NaCl, 10% glycerol) and lysed
by sonication. The supernatants, after centrifugation at 15 000 r/min for
20 min, containing soluble cytoplasmic proteins, were loaded onto 10 mL Ni-NTA
affinity column and eluted with NTA-20 (20 mmol/L Tris・HCl pH 7.9, 0.5 mol/L
NaCl, 10% glycerol, 20 mmol/L imidazole). Eluted fractions were monitored
for protein at A280, and the relevant fractions
were pooled and dialyzed to enzymatic active buffer (0.1 mol/L phosphate buffer,
pH 7.6, containing 0.1% Tween-20), subsequently, the sample was concentrated
to about 5 g/L by ultrafiltration (Milipore).
1. 4 Enzymatic digestion of the fusion proteins and RP-HPLC analysis of
purified C-peptide
Fusion multiple polymers including three copies of C-peptide (Fig.1), was
dissolved in 0.1 mmol/L phosphate buffer, pH 7.5, containing 0.1% Tween-20
to the protein concentration of 1.5 g/L. Trypsin (Dibco) and recombinant carboxypeptidase
B, were added to trypsin/fusion protein ratios of 1∶200(by mass) and carboxypeptidase
B /fusion protein ratios of 1∶100(by mass) respectively. After 30, 90 and
140 min respectively, samples were taken from the cleavage mixtures and analyzed
by RP-HPLC (250 mm Kromasil C8 column: inner diameter, 4.6 mm; particle size,
7 μm; pore size, 10 nm ), on a Hewlett Packard 1100 HPLC (Grenoble, France).
Elution was performed by using a 10%－40% acetonitrile gradient containing
0.1% trifluoroacetic acid for 30 min at 40 ℃, with a flow rate of 1 mL/min.
The A214 was monitored, and relevant fractions
of C-peptide were pooled and lyophilized.
1. 5 Characterization of the produced C-peptide with IMMULITE C-Peptide
and amino acid composition analysis
The lyophilized C-peptide was dissolved in 0.05 mol/L Na-phosphate buffer,
pH 7.4, its concentration was determined by the Bradford method［23］, using
bovine serum album as standard protein. The recombinant C-peptide was diluted
to final concentration within 0.5－20 μg/L with 0.05 mol/L Na-phosphate buffer,
pH 7.4, 10 g/L BSA, and was analyzed using a commercially available IMMULITE
C-peptide kit (DPC, USA; catalog number: LKPE5), which was developed to monitor
human C-peptide in serum, heparinized plasma, or urine. Briefly, the assay
involved barcode-labeled rabbit anti-C-peptide, alkaline phosphatase (bovine
calf intestine) conjugated C-peptide and human C-peptide standards (low and
high) in buffered human albumin.
Amino acid analysis was performed with about 50 μg lyophilized sample purified
with RP-HPLC. The result of amino acid composition was compared to that of
human C-peptide.
1. 6 Stability of recombinant proinsulin C-peptide in aqueous solution
Samples were prepared by adding 0.1 mL of 0.1 mol/L Na-titrate buffer (pH
3) or 0.1 mol/L Tris・HCl buffer (pH 7.4 or pH 9) into reagent vials containing
lyophilized C-peptide. The reaction vials were then placed into a constant
temperature incubator (37 ℃ or 70 ℃) respectively. Samples were periodically
removed from the incubator and analyzed with RP-HPLC directly. C-peptide and
its degradation products were eluted and detected at 214 nm. A linear gradient
elution was employed: 20%A-80%B to 40%A-60%B over 20 min. Mobile phase A was
a 0.1% TFA/acetonitrile solution and mobile B was 0.1% TFA/distilled water.
The injection volume was 40 μl and the flow rate was 1 mL/min.
2 Results and Discussion
2.1 DNA constructions
In order to get higher yields of C-peptide, vectors carrying one, two and
three C-peptide encoding gene fragments were constructed. Constructs containing
one or three inserts were further subcloned to yield the two expression vectors
pET-32a-cp1 and pET-32a-cp3, fusion protein includes 6×His tag, which enabled
efficient affinity purification on Ni-NTA affinity Sepharose column.
2.2 Production and affinity purification of the fusion protein
As shown in Fig.1, fusion proteins with one or three copies of C-peptide were
expressed by recombinant plasmids pET-cp1 and pET-cp3 in host cells HMS174
(DE3) at high level, and the apparent molecular weight of them were estimated
as approximately 23 kD and 31 kD, respectively. As expected, the yield of
C-peptide should be increased in proportion to its multiplicity, since the
two fusion proteins were produced at similar level (Fig.2), about 60－80 mg/L.

Fig.1 Schematic presentation
of the fusion protein amino acid sequence of the C-peptide
The TASQA linker region indicated in single letter code flanking the C-peptide
is derived from SfiI restriction sequence. Note that Arg residues (in
bold) flank each C-peptide.

Fig.2 SDS-PAGE analysis of fusion
expression of multiple C-peptides with 15% polyacrylamide under denature conditions
The samples of expressed products were analyzed by 15% polyacrylamide gel
under denature conditions and Coomassie blue staining. M, marker; 1 and 2,
proteins extracted from the cells HMS174(DE3) containing the expression vector
pET-cp1 without IPTG induction (1) and after IPTG induction (2); 3 and 4,
proteins extracted from the cells HMS174(DE3) containing the expression vector
pET-cp3 without IPTG induction (3) and after IPTG induction (4).
While C-peptide encoding gene was introduced to pT7－473 with a small fusion
6×His encoding gene, the fused protein could not be produced whatever denoted
in cells HMS174 (DE3) or BL21 (DE3) or BL21 (DE3) pLys. Perhaps the expressed
small peptide did harm to the host cells. The expressed vector pET-32a, which
contained large fusion protein gene was constructed, and the apparent molecular
weight of pure fusion protein itself was about 18 kD, in this way, C-peptide
was expressed successfully.
Ni-NTA affinity chromatography efficiently separated the expressed fusion
protein from the supernatant, resulting in above 70% purity of the fusion
protein about 37.5 mg/L (Fig. 3).
2.3 Enzymatic digestion of the fusion proteins and RP-HPLC analysis of
purified C-peptide
We have used trypsin in combination with carboxypeptidase B for the process
of fusion proteins in order to get native human C-peptide monomers. Trypsin
thus cleaved the fusion protein C-terminally of each Arg residue (Fig.3),
and carboxypeptidase B removed the C-terminal Arg residue presented on each
C-peptide after trypsin digestion to get native C-peptide (Fig.3). To investigate
when the trypsin+recombinant carboxypeptidase B treatment reached completion,
the fusion protein was subjected to enzymatic processing for 30, 90 and 140
min respectively (data not shown), and it was concluded that fusion protein
was completely processed after 140 min treatment (Fig. 3).

Fig.3 RP-HPLC analysis of the
purified fusion protein (A) and enzymatic cleavage mixtures with trypsin single
(B), trypsin+CPB(recombinant) for 140 min (C) and comparative standard C-peptide
(D), respectively
1, C-peptide; 2, C-peptide with an Arg residue on C-terminal; 3,fusion protein.
2.4 Characterization of the produced C-peptide
Three different analysis were performed in order to assess whether the obtained
was peptide corresponded to an authentic native human C-peptide. First, a
RP-HPLC analysis was used for comparison of the recombinant C-peptide to C-peptide
standard and both retention time were found to be identical (Fig. 3). Second,
the recombinant C-peptide was analyzed using a commercially available IMMULITE
C-Peptide, which was for the quantitative measurement of C-peptide in serum,
heparinized plasma, or urine. It was well quantified as 14.9 mg/L, compared
to 17 mg/L with Bradford［23］ method to measure total protein concentration.
Third, the recombinant C-peptide was subjected to amino acids composition
analysis, and the result was identical to the amino acids composition of human
C-peptide. The good agreement of amino acids composition, together with shown
similarities of the recombinant C-peptide to C-peptide standard in the comparative
RP-HPLC analysis (Fig.3) and IMMULITE C-Peptide quantitative assay, indeed
suggested that the recombinant C-peptide obtained in this report was the native
human C-peptide.
2.5 Stability of recombinant proinsulin C-peptide in aqueous solution
C-peptide was a polypeptide comprised of 31 amino acid residues arranged on
a single chain. There were five acid amino acid residues and no alkaline amino
acid residues (Fig.1), four Glu residues spread around the chain and one Asp
residue was adjacent to N-terminal. Hydrolysis could take place at either
the N-terminal and /or C-terminal peptide bonds adjacent to the Asp residue.
The mechanism of hydrolysis undoubtedly involved intramolecular catalysis
by a carboxyl group of the Asp residue in acidic media［24，26］. Ser residue
could also undergo beta-elimination at alkaline conditions. In many cases,
the beta-elimination reaction was influenced by pH and temperature. Although
C-peptide could undergo degradation via a variety of chemical reactions including
beta-elimination, racemization and oxidation, which were specific to certain
amino acid residues, deamination of C-terminal of polypeptide was the most
common chemical pathway of polypeptide degradation under alkaline pH［24－26］.

With the exception of the reduced recombinant proinsulin C-peptide as peak
1, the degradation products had not been identified in this study. The degradation
products might be a form of C-peptide deamidated at the end of C-terminal
of C-peptide at pH 9［27］.
The C-peptide degradation showed a marked dependence on pH and temperature.
Figures also suggested that C-peptide might degrade by several different specific
pathways at pH 3 and pH 9 (Fig. 4). It was found that the pH 3 affected the
degradation rate of C-peptide and that the observed degradation products increased
as the degradation of C-peptide progressed and the degradation products were
complicated at 70 ℃ for 10 h (Fig.5). While the major degradation products
at pH9 appeared to be relatively stable to temperature (Fig.5， Fig.6). No
C-peptide degradation products were detected at pH 7.4 under 37 ℃ for 10 h
and pH 7.4 under 70 ℃ for 3 h while 10 g/L BSA existed (Fig.5, Fig.6). 99%
and 96% of total C-peptide remained at pH 7.4 with and without 10 g/L BSA
under 37 ℃ for 10 h respectively (Fig.6). �

Fig.4 RP-HPLC analysis of C-peptide
prepared in different pH buffers
(A) The collected C-peptide from fusion proteins enzymatic cleavage mixture
before lyophilized. (B)－(E)， The lyophilized C-peptide was prepared in different
pH buffers respectively and analyzed immediately. (B) pH 7.4, 0.1 mol/L Tris・HCl
buffer containing 10 g/L BSA; (C) pH 3, 0.1 mol/L Na-titrate buffer; (D) pH
7.4, 0.1 mol/L Tris・HCl buffer; (E) pH 9.0, 0.1 mol/L Tris・HCl buffer. 1,
recombinant C-peptide; other peaks, unknown.

Fig.5 RP-HPLC analysis of degradation
of C-peptide prepared in different pH buffers kept at 70 ℃
(A) pH 7.4, 0.1 mol/L Tris・HCl buffer containing 10 g/L BSA, 3 h; (B) pH 7.4,
0.1 mol/L Tris・HCl buffer, 6 h; (C) pH 3.0, 0.1 mol/L Na-titrate buffer, 10
h; (D) pH 9.0, 0.1 mol/L Tris・HCl buffer, 10 h. 1, recombinant C-peptide；
other peaks, unknown.

Fig.6 RP-HPLC analysis of degradation
of C-peptide prepared in different pH buffers kept at 37 ℃ for 10 h
(A) pH 7.4, 0.1 mol/L Tris・HCl buffer containing 10 g/L BSA; (B) pH 7.4, 0.1
mol/L Tris・HCl buffer; (C) pH 3.0, 0.1 mol/L Na-titrate buffer; (D) pH 9.0,
0.1 mol/L Tris・HCl buffer. 1, recombinant C-peptide； other peaks, unknown.

The degradation kinetics of C-peptide was also studied, Fig.7 showed a first-order
plot of the residual percentage amounts of C-peptide vs. time in various pH
solution at 37 ℃ or 70 ℃. It was found that pH affected the degradation first
(Fig.4). At the same pH, the temperature affected the degradation rate of
C-peptide and the observed degradation reaction rates approximately followed
first-order kinetics (Fig.7).

Fig.7 First-order plot for the
degradation of recombinant C-peptide in 0.1 mol/L Na-titrate buffer (pH 3)
and 0.1 mol/L Tris・HCl buffer (pH 7.4 or pH 9) at 37 ℃or 70 ℃Conclusively,
we had successfully constructed gene carrying three copies of a C-peptide
encoding gene fragments, which had been expressed in E. coli as a fusion
protein at high level, about 80 mg/L. Ni-NTA affinity chromatography efficiently
separated the expressed fusion protein from the supernatant. Native C-peptide
was obtained by trypsin+carboxypeptidase B treatment of fusion protein. The
good agreement of amino acids composition, together with shown similarities
of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC
analysis and IMMULITE C-Peptide quantitative assay, indeed suggested that
the recombinant C-peptide obtained in this report was the native human C-peptide.
Recombinant C-peptide was stable in pH 7.4 buffered solution, 10 g/L BSA showed
protect effect to it in pH 7.4 buffered solution. The activities of proinsulin
C-peptide were being investigated in our laboratory.

�お�
Acknowledgements We thank Professor SUN Xiang-Ming and Dr. XIA Wen-Chao
for excellent assistance with RP-HPLC analysis, Dr. GU for assistance with
IMMULITE C-Peptide quantitative assay in Nuclear Medical Laboratory of Zhongshan
Hospital, and we are also grateful to Dr. ZHANG Hui-Tang, Dr. YAN Zhi-Qiang,
Dr. CHEN Yan, Dr. SHEN Yan and Dr. YU Gu-Song for fruitful discussion and
kind support.

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Received: June 24, 2003Accepted:
August 18, 2003
*Corresponding authors: YUAN Qin-Sheng: Tel, 86-21-64252255；Fax, 86-21-64252255；e-mail,
[email protected]
GONG-Yi: Tel, 86-21-64700892-369; Fax, 86-21-64700244； e-mail, [email protected]

 

Updated at: 12-18-2003