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Acta Biochim Biophys Sin 2006, 38: 586-592

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 poly�nucleotide 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 �C 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'-GGAATTCATGCATCATCA�TCATC�ATCATAGATCTAAGGAAGCTGAAGAT-3' and 5'-GGAT�CCAGCCTTTTGCAAAGAACCTTCCAAAG-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 �C 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 �C. 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 �C 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%).

 

 

References

 

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