Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00200.x |
Human Proinsulin C-peptide
from a Precursor Overexpressed in Pichia pastoris
Yang-Bin HUANG2#,
Jiang LI1#, Xin GAO3,
Jiu-Ru SUN4, Yi LU5,
Tao FENG2, Jian FEI1,
Da-Fu CUI1, Qi-Chang XIA1,
Jun REN2*, and You-Shang Zhang1*
1 Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China;
2
Shanghai Newsummit Biopharma Company, Shanghai 200233, China;
3 Zhongshan Hospital, Fudan University,
Shanghai 200031, China;
4 Shanghai Yizhong Biotechnology Company,
Shanghai 201203, China;
5 Shanghai Center of Research and
Development of New Drugs, Shanghai 201203, China
Received: April 6,
2006
Accepted: May 23,
2006
#
These
authors contributed equally to this work
*Corresponding
authors:
You-shang ZHANG: Tel, 86-21-54920237; Fax,
86-21-54921011; E-mail, [email protected]
Jun REN: Tel, 86-21-50798788;
Fax, 86-21-50788766; E-mail, [email protected]
Abstract In this article we report the production
of human proinsulin C-peptide with 31 amino acid residues from a precursor
overexpressed in Pichia pastoris. A C-peptide precursor expression
plasmid containing nine C-peptide genes in tandem was constructed and used to
transform P. pastoris. Transformants with a high copy number of the
C-peptide precursor gene integrated into the chromosome of P. pastoris
were selected. In high-density fermentation in a 300 liter fermentor using a
simple culture medium composed mainly of salt and methanol, the C-peptide
precursor was overexpressed to a level of 2.28 g per liter. A simple procedure
was established to purify the expression product from the culture medium. The
purified C-peptide precursor was converted into C-peptide by trypsin and
carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96%
was 730 mg per liter of culture. The purified C-peptide was characterized by
mass spectrometry, N- and C-terminal amino acid sequencing, and sodium
dodecylsulfate-polyacrylamide gel electrophoresis.
Key words proinsulin; C-peptide; Pichia pastoris
The incidence of diabetes is increasing rapidly. At present,
approximately 150 million people suffer from this disease worldwide. In
diabetic patients, various complications usually occur even with
well-controlled blood glucose levels. No efficient medicine is currently
available to prevent diabetic complications.
In pancreatic b-cells, C-peptide with 31 amino acid residues is cleaved off from
proinsulin and co-secreted with insulin in response to glucose stimulation.
Formerly, C-peptide was considered to possess no biological function other than
its role in insulin biosynthesis. Recently, it was reported that C-peptide
could ameliorate sensory nerve dysfunction, nephropathy, neuropathy [1,2], and
impaired myocardial function [3] in patients with type I diabetes lacking
endogenous C-peptide. In diabetic rats, C-peptide could prevent vascular and
neural dysfunction, nephropathy, glomerular hypertrophy, albuminuria,
glomerular hyperfiltration and hippocampal apoptosis [4–7]. It was also reported
that C-peptide could stimulate rat renal tubular Na+-K+-ATPase activity [8]. These results suggest that C-peptide might be
a new medicine to prevent diabetic complications. For pre-clinical and clinical
studies, it is necessary to produce human C-peptide on a large scale. It is
difficult to express a single C-peptide because of its low molecular weight and
its random structure in solution. One strategy is to express it in the form of
fusion protein. Another strategy is to express a gene encoding multiple copies
of the small peptide [9,10] including the expression of C-peptide in Escherichia
coli [11].
Here we report the expression of tandemly repeated C-peptide in Pichia
pastoris and the enzymatic cleavage of the purified expression product to
yield human C-peptide.
Materials and Methods
Construction of plasmid
p36DD/PIC9 to express C-peptide precursor
The flow sheet of plasmid construction is shown in Fig. 1. A
double-stranded C-peptide gene of the sequence encoding C-peptide and an extra
C-terminal Lys with 5‘ BglII site and 3‘ BamHI site
was constructed from six oligonucleotides (Fig. 2).
Each oligonucleotide (100 pmol) was phosphorylated in 10 ml reaction
mixture containing 0.5 ml T4 polynucleotide kinase (New England Biolabs, Ipswich, USA), 10
nmol ATP and 1 ml buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM
dithiothreitol). One microliter of each phosphorylated oligonucleotide solution
was mixed and incubated at 70 ºC for 10 min with subsequent cooling to room
temperature. T4 DNA ligase (New England Biolabs) was added and the mixture was
incubated at room temperature for 2 h. The double-stranded DNA containing the
C-peptide gene was ligated with pCR-Blunt (Invitrogen, Carlsbad, USA) to
obtain p32/ZB. As 5‘ BglII and 3‘ BamHI in the
C-peptide gene are isocaudarners, they were used to produce multiple copies of
the C-peptide gene. The C-peptide gene in p32/ZB was digested with BglII/BamHI
(New England Biolabs) and joined by T4 DNA ligase to obtain multicopy C-peptide
gene, followed by BglII/BamHI digestion to cleave ligated product
with wrong direction. The multicopy C-peptide gene was inserted into p32/ZB,
which was linearized by BglII and digested with bovine small intestine
phosphatase (New England Biolabs) to remove the 5‘ phosphate group.
p32DD/ZB containing the largest number of C-peptide gene was selected by PCR
using T7 promoter and M13 reverse as primers. To facilitate characterization
and purification, a His6 tag was added to the C-peptide
gene. PCR catalyzed by Pfu DNA polymerase (Promega, Madison, USA) was carried
out using p32/ZB as the template and 5‘-GGAATTCATGCATCATCATCATCATCATAGATCTAAGGAAGCTGAAGAT-3‘
and 5‘-GGATCCAGCCTTTTGCAAAGAACCTTCCAAAG-3‘ as the primers. The
PCR product was ligated with pCR-Blunt to obtain p36/ZB. BglII and BamHI
digested fragment was cleaved from p32DD/ZB and inserted into the BglII
site of p36/ZB to obtain p36DD/ZB. A 1 kb DNA fragment containing the C-peptide
tandem gene was cleaved from p36DD/ZB by EcoRI (New England Biolabs)
digestion and inserted into the EcoRI site of pPIC9 to obtain the
expression plasmid p36DD/PIC9. p36DD/PIC9 was confirmed by DNA sequencing and
the C-peptide gene number was determined by 1.2% agarose gel electrophoresis
after EcoRI digestion.
Transformation of P.
pastoris by p36DD/PIC9
p36DD/PIC9 was linearized with SalI and cloned into P.
pastoris GS115 (his4; Invitrogen) by electroporation. The His+ transformants were grown in YPD medium (1% yeast extract, 2%
peptone, 2% dextrose). The chromosome DNA was extracted and spotted on a nylon
membrane. The high expression transformant C-peptide/pPIC9 with a high copy
number of the C-peptide precursor gene integrated into the chromosome was
selected using the dot blotting method [12] using the C-peptide tandem gene as
the probe.
Shake flask culture of
transformant C-peptide/pPIC9
Transformant C-peptide/pPIC9 was grown in 20 ml YPD medium at 30 ºC
for 2 d. The cells were collected by centrifugation, resuspended in 10 ml YPM
medium (YPD medium with dextrose replaced by methanol) and grown for 3 d. After
centrifugation, 10 ml supernatant was analyzed by SDS-PAGE and Western blot using mouse
anti-C-peptide antibody (Dakocytomation, Glostrup, Denmark) as the first
antibody and horseradish peroxidase-conjugated rabbit anti-mouse antibody
(Sino-American Biotech, Shanghai, China) as the second antibody.
High density fermentation
High density fermentation was carried out basically according to Pichia
protocols [13]. The following media were used. Each liter of the basal salt medium
(BSM) contained 40 g glycerol, 27 ml H3PO4, 1 g CaSO4∙2H2O, 18 g
K2SO4, 7 g MgSO4, 1.68 g
sodium citrate∙2H2O and 4.0 g KOH. Each liter of
the trace element solution (PTM1) contained 6 g CuSO4, 0.08 g
KI, 3 g MnSO4∙H2O, 0.2 g Na2MoO4∙2H2O, 0.02 g H3BO3, 20 g ZnCl2, 5 ml H2SO4, 65 g FeSO4∙7H2O, 0.5 g CoCl2∙2H2O and 0.2 g biotin. PTM1 solution was sterilized by filtration. One
liter of BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 10% yeast
nitrogen base, 0.2% biotin, 0.1 M potassium phosphate buffer, pH 6.0) in a
flask was inoculated and cultured for 20 h at 30 ºC. The culture was
transferred to 11 liters of BSM medium containing 44 ml PTM1 in a 20 liter
fermentor (Bioengineering, Wald, Switzerland) and cultured for 4 h. The
culture was added to 100 liters of BSM medium containing 400 ml PTM1 in a 300
liter fermentor
(Bioengineering). The pH was adjusted to and maintained at 5 with 30% NH4OH. After approximately 20–24 h, when glycerol was depleted, 50% glycerol
containing PTM1 (12 ml per liter of 50% glycerol) was fed for 4–8 h, followed by
24–48 h methanol feeding (methanol with 12 ml PTM1 per liter of
methanol). The supernatant was collected by centrifugation.
Purification of C-peptide
precursor by hydrophobic chromatography
NaCl was added to the supernatant to a concentration of 3 M. The
solution was applied to a phenyl Sepharose FF (Amersham Pharmacia Biotech,
Piscataway, USA) hydrophobic chromatography column and washed with 20 mM
phosphate buffer (pH 7.4) containing 3 M NaCl to remove impurities. The
C-peptide precursor was eluted from the column with 20 mM phosphate buffer (pH
7.4) at a flow rate of 60 cm/h for 0.5 column volume.
Enzymatic digestion of
C-peptide precursor
The eluted C-peptide precursor at a concentration of approximately
9.5 mg/ml, determined by the Lowry method [14], was digested with trypsin
(Sigma-Aldrich, St. Louis, USA) at an enzyme-to-substrate ratio of 1:600 by
weight, and carboxypeptidase B (Worthington, Lakewood, USA) at an
enzyme-to-substrate ratio of 1:1500 by weight at 30 ºC in the presence of 1 mM
CaCl2. Samples were taken at a 1 h interval, adjusted to pH 2–3 by adding HCl,
and the enzymatic digestion of C-peptide precursor was monitored by HPLC using
a 250 mm Ultrasil C18 column (Beckman, Fullerton, USA) (inner diameter 4.6 mm,
particle size 10 mm, pore size 10 nm) and a 2487 HPLC instrument (Waters, Detroit,
USA). The column was eluted with 21%–42% (v/v) acetonitrile gradient
containing 0.08% (v/v) trifluoroacetic acid at a
flow rate of 1 ml/min. The A230 was measured.
Purification of C-peptide
The digested product was purified by ultrafiltration. The solution
retained by the 10 kDa film was equilibrated with 10 mM phosphate buffer (pH 7.4),
applied to a Sepharose QFF column (Amersham Pharmacia Biotech) and washed with
10 mM phosphate buffer (pH 7.4) to remove impurities. C-peptide was eluted from
the column with the same buffer containing 1 M NaCl at a flow rate of 30 cm/h
for two column volume. The A230 was measured. The
concentration of C-peptide was determined by HPLC using synthetic C-peptide as
standard.
Characterization of C-peptide
The C-peptide was identified and characterized by mass spectrometry
(Finnigan LCQ; Thermo Electron, San Jose, USA), N- and C-terminal amino acid
sequencing (PE-ABI 491A; Applied Biosystems, Foster, USA) and tricine-SDS-PAGE.
Results
Cloning and expression of
C-peptide precursor
DNA sequencing and agarose gel electrophoresis of EcoRI-digested
p36DD/PIC9 (Fig. 3) showed that the C-peptide precursor contained nine
copies of C-peptide. A transformant with a high copy number of p36DD/PIC9 was
selected by dot blotting (Fig. 4).
The expression level of C-peptide precursor in the shake flask
estimated by SDS-PAGE reached 100 mg per liter culture (Fig. 5). In the
high density fermentation using the 300 liter fermentor, the expression level
of C-peptide precursor increased with prolonged methanol induction time, and
reached 2.28 g per liter culture after 48 h methanol induction as shown by the
SDS-PAGE (Fig. 6). The western
blot analysis results of C-peptide precursor expressed in the 300 liter
fermentor are shown in Fig. 7.
Purification of C-peptide
precursor
The expressed C-peptide precursor was purified by hydrophobic
chromatography that has the advantage of concentrating the precursor and
removing the impurities at the same time. The result is shown in Table 1.
Enzymatic digestion of
purified C-peptide precursor
The digest of purified C-peptide precursor by trypsin and
carboxypeptidase B was analyzed by HPLC, and the result indicated that the
precursor was completely digested after 3 h (Fig. 8).
Purification of C-peptide
C-peptide was purified by ultrafiltration and Sepharose QFF chromatography.
The result is shown in Table 2. C-peptide preparation was analyzed by
HPLC before and after Sepharose QFF chromatography (Fig. 9). The overall
yield of C-peptide preparation with a purity of 96% was 73 g per 100 liters of
culture.
Purified C-peptide was homogeneous in HPLC. Its molecular mass
determined by mass spectrometry was 3020.0 Da, in agreement with the
theoretical value of 3020.3 Da. N- and C-terminal amino acid sequencing showed
its amino acid sequence as E-A-E-D-L-Q-V-G-Q-V-E-L-G-G-G-P-G-A-G-S-L-Q-P-L-A-L-E-G-S-L-Q
and C-terminal sequence as Q-L-S-G-E. Amino acid analysis showed that the
purified C-peptide had the expected amino acid composition (data not shown).
Discussion
Proinsulin as a precursor in insulin biosynthesis was first
discovered by Steiner et al. [15]. Subsequently, proinsulin was
isolated from crystalline porcine insulin preparation and its amino acid
sequence was determined by Chance et al. [16]. The C-peptide connecting
the B and A chains of insulin will naturally facilitate the right pairing of
disulfide bonds in insulin. At first, C-peptide was thought to have no other
physiological functions, even though it is hard to imagine why such a long
connecting peptide of 31 residues, instead of a short spacer, would be needed
to join the C-terminus of the B chain and the N-terminus of the A chain when
they are so closely located. In 1997, the prevention of vascular and neural
dysfunction in diabetic rats by C-peptide was reported by Eli Lilly Company
[4]. Since then, more and more experimental and clinical studies on the
beneficial effects of C-peptide on diabetes have been reported. For further
pre-clinical and clinical studies, it is essential to have a large amount of
human C-peptide. In expressing C-peptide with a random solution structure and
easily degraded in vivo, we adopted the strategy of joining nine
C-peptide genes in tandem to form the C-peptide precursor gene. The recombinant
C-peptide precursor was easily converted into C-peptide in vitro by
joint trypsin and carboxypeptidase B digestion. The expression system we used
is P. pastoris which gave high expression of many proteins, including
insulin, in high density fermentation. The C-peptide precursor expressed in
shaken flask was shown by Western blot as a single band (data not shown).
However, in high density fermentation, in addition to C-peptide precursor as
the main component, degraded fragments were also present, as shown by Western
blot (Fig. 7). Fortunately, the degradation by trypsin-like digestion
occurred after Lys but not in C-peptide, so the degraded products are fragments
containing different numbers of C-peptide. These fragments pooled together
were easily converted into C-peptide in vitro by joint trypsin and
carboxypeptidase B digestion. A His6 tag was added to the
C-peptide gene to facilitate characterization and purification. However, it
was found later that the His6 tag was not very efficient for
purifying C-peptide precursor, so it was only used to detect C-peptide
precursor by Western blot. The overexpressed C-peptide precursor secreted into
the cultural medium with a purity of 38% could be easily purified to 86% purity
by a single step of hydrophobic chromatography. After enzyme digestion, the
C-peptide with a purity of 96% was obtained by ultrafiltration and ion
exchange chromatography.
In summary, by using a combination of different effective measures,
including the joining of the C-peptide gene in tandem, screening of recombinant
P. pastoris with a high copy number of the integrated C-peptide
precursor gene, and expression of the secreted C-peptide precursor into a
simple culture medium in high density fermentation, a high expression level of
2.28 g C-peptide precursor per liter of culture was achieved in a 300 liter
fermentor, resulting in a high yield of highly purified C-peptide (73 g per
100 liters of culture with a purity of 96%).
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