生 物 化 学 与 生 物 物 理 学 报
ISSN 058229879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003 , 35 (11) : 986 - 992
CN 3121300/ Q
Expression of C2peptide Multiple Gene Copies in Escherichia coli
and Stabilities of C2peptide in Aqueous Solution
L I Su2Xia1 ,2 , TIAN Li2Ping2 , L IU Hai2Feng1 , ZHAN G Yu2J ian2 ,
2 2 1
HU Xiao2Bo, GON G Yi3 , YUAN Qin2Sheng3 ( 1 State Key L aboratory of Bioreactor Engineering , East China U niversity of Science and Technology , S hanghai 200237 , China ;
2
Research Center of Biotechnology , S hanghai Instit utes f or Biological Sciences , the Chinese Academy of Sciences , S hanghai 200233 , China )
Abstract A gene f ragment encoding three copies of proinsulin C2peptide was synthesized and expressed in E. coli and the recombinant proinsulin C2peptide was produced through site2specific 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. Ni2N TA affinity chromatography efficiently separated the expressed fusion protein f rom the supernatant , to obtain about 37. 5 mg/ L of the fusion protein with 70 % purity. Enzymatic digestion by t rypsin and carboxypeptidase B of the fusion protein efficiently released native C2peptide , the overall yield of recombinant C2peptide at a purity over 95 % was 1. 5 mg/L . The good agreement of amino acids composition , together with shown similarities of the recombinant C2peptide to C2peptide standard in the comparative RP2HPL C analysis and IMMUL ITE C2Peptide quantitative assay , suggested that the recombinant C2peptide obtained in this report was the native human C2peptide. The investigation of the chemical stability of recombinant human C2peptide in aqueous solutions by RP2HPL C was also reported. The degradation of the recombinant C2peptide showed a marked dependence on p H and temperature. The degradation reaction of C2peptide occurred immediately in p H 3 or p H 9 buffered solution. The degradation reaction of C2peptide followed first2 order kinetics in p H 3 buffered solution at 37 ℃or 70 ℃, only 40. 3 % of C2peptide was remained after 10 h at 70 ℃. The maximum stability was achieved at p H 7. 4 , more than 90 % of C2peptide were detected at p H 7. 4 and 37 ℃after 10 h and at p H 7. 4 and 70 ℃after 5 h. 99 % and 96 % of C2 peptide was remained at p H 7. 4 and 37 ℃after 10 h with and without 10 g/ L BSA respectively.
Key words proinsulin C2peptide ; multiple copies ; expression ; stability ; RP2HPL C
The proinsulin C2peptide had long been considered a by2p roduct of insulin biosynthesis , and it was important in the folding of proinsulin by providing a spacer sequence that could be removed
[ 1 ,2 ]
once folding was completed. After the discovery of the model of insulin biosynthesis , several early studies addressed the question of possible physiological effects of C2peptide , for example , insulin2like 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
Received : J une 24 , 2003 Accepted : August 18 , 2003 3 Corresponding authors :
YUAN Qin2Sheng : Tel , 86221264252255 ; Fax , 86221264252255 ; e2mail,****************.cn
GON G2Yi : Tel , 862212647008922369 ; Fax , 86221264700244 ; e2 mail,**************.cn
C2peptide to the plasma membranes of intact cells and to detergent2solubilized cells had been shown , which indicated the existence of a cell surface receptor for C2
[ 5 ]
peptide. Data now indicated that C2peptide in the nanomolar concentration range bound specifically to cell surfaces , probably to a G protein2coupled membrane receptors , with subsequent activation of Ca2 + 2dependent intracellular signaling pathways[ 6 ,7 ] . The binding was stereospecific , and no cross2reaction was seen with insulin , proinsulin , insulin growth
[ 8 ]
factors I and II , or neuropeptide Y. C2peptide elicited a number of cellular responses , including 2 + [ 9 ] Cainflux, activation of mitogen2activated
[ 10 ] + +
protein ( MAP) kinase, activation of Na , K2
[ 11 ] [ 12 ]
A TPaseand endothelial NO synthase. Data also indicated that C2peptide administration was accompanied by augmented blood flow in skeletal
[ 13 ]
muscle and skin, diminished glomerular hyperfiltration , reduced urinary albumin excretion[ 14 ] , and improved nerve function[ 15 ] , in all
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987
patients with type 1 diabetes who lacked C2peptide , but not in healthy subjects. The possibility existed that C2peptide replacement , together with insulin administration , might prevent the development or retard the progression of long2term complication in C2 peptide2deficient type 1 diabetes patients[ 16 ] .
Production st rategies for recombinant peptides constituting small portions of larger fusion proteins generally gave relatively low yields of the target peptide. In the presented process , C2peptide constituted only 19 % of the fusion protein. In order to increase the amount of produced target peptide , one st rategy was to synthesize a gene product with [ 17 ] multiple copies of the target peptide. Multimerization of gene f ragments could be achieved by a number of methods. A technique for the polymerization and assembly of peptides involved the use of the class2IIs restriction enzyme BspM I , which enabled unidirectional insertion of the DNA f ragments
[ 18 ]
to be polymerizated. Two identical class2IIs 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 high2copy2number multimers of DNA f ragments in a tandem unite and up to 108 copies
[ 19 ]
were constructed. Head2to2tail polymerizations of synthetic DNA f ragments had been used in several reports to obtain gene multimerization by design of single2stranded non2palindromic ends. The DNA f ragments were polymerized and could then be directly ligated to matching protrusions resulted f rom
[ 20 ]
restriction enzyme digestion. Alternative st rategies involved polymerization of the gene construct and ligation of adapter molecules containing
[ 21 ]
restriction sites to allow further subcloning. 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 f ree 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 [ 22 ]
et al . , multiple copies of proinsulin C2peptide was obtained through introducing the restricted enzymatic site , Sf i I , to ensure the multiple C2peptide2encoding gene ligated head2to2tail. Expression vector p ET232a carrying one or three C2peptide2encoding gene fragments were constructed and were expressed at high level in E. coli as a fusion protein , C2peptide was recovered by trypsin + carboxypeptidase B enzymatically digestion and further purification with RP2HPLC.
There was few fundamental information on the chemical stability of C2peptide. This note described the pattern of degradation of C2peptide as a function of p H and temperature by RP2HPLC.
1 Materials and Methods
1. 1 Materials
All primers were synthesized by Bioasia Co. Ltd. ; restriction enzyme Sf i I was obtained from NewEngland Biolabs; general vector pMD182T , restricted enzymes EcoRI and HindIII , ligation kit and CIAP were purchased from Ta KaRa ( Dalian ) Co. Ltd. ;
R
IMMUL ITEC2Peptide 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
[ 22 ]
Partly based on a strategy of Jonasson et al . , briefly , two single strands DNA , partly complementary to each other ( indicated underline ) , were synthesized according to the bias for preferred codons of E. coli , C2p s ( 84 bp) : 5′2CCG GAA TTC CAG GCC TCC CAG GCC CGC GAA GCT GA G GAC CTG CAG GTT GGT CAG GTT GAA CTG GGC GGT GGC CCG GGT GCA GGC2 3′( containing EcoRI and Sf i I restricted enzyme sites at 5′end and arginine2encoding gene indicated with boxed) ; C2p a ( 77 bp) : 3′2C CCA C GT CC G A GA AAC GTC GGC GAC CGA AAC CTT CCA A GA GAA GTC GCA TGC CGGAGG GTC CGG TAA TTT CGA A GC G25′ ( containing Sf i I and HindIII restricted enzyme sites at 5′ end and arginine2encoding gene indicated with boxed ) . Two ssDNA gene fragments ( per 20 μg) was allowed to extend and anneal in the
presence of 1 u Pyrobest polymerase ( Ta KaRa ) 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 pMD182T , the ligation mixture was transformed to the dcm2 E. coli strain JM110 to allow efficient Sf i I digestion. The purified C2peptide2encoding gene fragment was allowed to polymerized in a head2to2tail fashion , and were then ligated to the purified Sf i I digested plasmid which had been dephosphorylated with CIAP ( calf intestinal alkaline phosphatase) to prevent self2ligation. E. coli JM105 cells were transformed with the ligation mixture , and the resulting transformants were screened using a PCR2screening technique. Gene fragments encoding one , two and three concatamerized C2peptides were isolated and ligated to expression vector p ET232a through EcoRI and HindIII enzymatic sites , and the resulting plasmids were denoted p ET232a2cp1 , p ET232a2 cp2 and p ET232a2cp3 , respectively.
1. 3 Production and aff inity purif ication of the fusion protein
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The expression vectors were transformed into
E. coli HMS174 (DE3) , the cells harboring the desired expression vectors were grown individually for a pre2
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 A 600 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 N TA20 ( 20 mmol/ L Tris·HCl p H 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 Ni2 N TA affinity column and eluted with N TA220 ( 20 mmol/ L Tris·HCl p H 7. 9 , 0. 5 mol/ L NaCl , 10 % glycerol , 20 mmol/ L imidazole) . Eluted fractions were monitored for protein at A 280 , and the relevant fractions were pooled and dialyzed to enzymatic active buffer (0. 1 mol/ L phosphate buffer , p H 7. 6 , containing 0. 1 % Tween220) , subsequently , the sample was concentrated to about 5 g/ L by ultrafiltration (Milipore) .
1. 4 Enzymatic digestion of the fusion proteins and RP2 HPLC analysis of purif ied C2peptide
Fusion multiple polymers including three copies of C2 peptide ( Fig. 1) , was dissolved in 0. 1 mmol/ L phosphate buffer , p H 7. 5 , containing 0. 1 % Tween220 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 RP2HPLC (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 A 214 was monitored , and relevant fractions of C2peptide were pooled and lyophilized.
1. 5 Characterization of the produced C2peptide with IMMUL ITE C2Peptide and amino acid composition analysis
The lyophilized C2peptide was dissolved in 0. 05 mol/ L Na2phosphate buffer , p H 7. 4 , its concentration
[ 23 ]
was determined by the Bradford method, using bovine serum album as standard protein. The recombinant C2 peptide was diluted to final concentration within 0. 5 - 20 μg/ L with 0. 05 mol/ L Na2phosphate buffer , p H 7. 4 , 10 g/ L BSA , and was analyzed using a commercially available IMMUL ITE C2peptide kit ( DPC , USA ; catalog number : L KPE5 ) , which was developed to monitor human C2peptide in serum , heparinized plasma , or urine. Briefly , the assay involved barcode2labeled
rabbit anti2C2peptide , alkaline phosphatase ( bovine calf intestine) conjugated C2peptide and human C2peptide standards (low and high) in buffered human albumin.
Amino acid analysis was performed with about 50μg lyophilized sample purified with RP2HPLC. The result of amino acid composition was compared to that of human C2 peptide.
1. 6 Stability of recombinant proinsulin C2peptide in aqueous solution
Samples were prepared by adding 0. 1 mL of 0. 1 mol/ L Na2titrate buffer (p H 3) or 0. 1 mol/ L Tris·HCl buffer (p H 7. 4 or p H 9) into reagent vials containing lyophilized C2peptide. 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 RP2HPLC directly. C2 peptide and its degradation products were eluted and detected at 214 nm. A linear gradient elution was employed : 20 %A280 %B to 40 %A260 %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 40μl and the flow rate was 1 mL/ min.
2 Results and Discussion
2. 1 DNA constructions
In order to get higher yields of C2peptide , vectors carrying one , two and three C2peptide encoding gene fragments were constructed. Constructs containing one or three inserts were further subcloned to yield the two expression vectors p ET232a2cp1 and p ET232a2cp3 , fusion protein includes 6 ×His tag , which enabled efficient affinity purification on Ni2N TA affinity Sepharose column.
2. 2 Production and aff inity purif ication of the fusion protein
As shown in Fig. 1 , fusion proteins with one or three copies of C2peptide were expressed by recombinant plasmids p ET2cp1 and p ET2cp3 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 C2peptide 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.
While C2peptide encoding gene was introduced to p T7 - 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 , p ET232a , 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 , C2peptide was expressed successfully.
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Fig. 1 Schematic presentation of the fusion protein amino acid sequence of the C2peptide
The TASQA linker region indicated in single letter code flanking the C2
peptide is derived from Sf i I restriction sequence. Note that Arg residues (in bold) flank each C2peptide.
Fig. 2 SDS2PAGE analysis of fusion expression of multiple C2 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 p ET2cp1 without IPTGinduction (1) and after IPTGinduction (2) ; 3 and 4 , proteins extracted from the cells HMS174 ( DE3) containing the expression vector p ET2cp3 without IPTG induction ( 3 ) and after IPTG induction (4) .
Fig. 3 RP2HPLC analysis of the purif ied fusion protein ( A) and enzymatic cleavage mixtures with trypsin single ( B) , trypsin + CPB( recombinant) for 140 min ( C) and comparative standard C2 peptide ( D) , respectively
1 , C2peptide ; 2 , C2peptide with an Arg residue on C2terminal ; 3 ,fusion
protein.
Ni2N TA 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 RP2 HPLC analysis of purif ied C2peptide
We have used trypsin in combination with carboxypeptidase B for the process of fusion proteins in order to get native human C2peptide monomers. Trypsin thus cleaved the fusion protein C2terminally of each Arg residue ( Fig. 3) , and carboxypeptidase B removed the C2 terminal Arg residue presented on each C2peptide after trypsin digestion to get native C2peptide ( 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) .
2. 4 Characterization of the produced C2peptide
Three different analysis were performed in order to assess whether the obtained was peptide corresponded to an authentic native human C2peptide. First , a RP2HPLC analysis was used for comparison of the recombinant C2
peptide to C2peptide standard and both retention time were found to be identical ( Fig. 3 ) . Second , the recombinant C2peptide was analyzed using a commercially available IMMUL ITE C2Peptide , which was for the quantitative measurement of C2peptide in serum , heparinized plasma , or urine. It was well quantified as
[ 23 ]
14. 9 mg/ L , compared to 17 mg/ L with Bradfordmethod to measure total protein concentration. Third , the recombinant C2peptide was subjected to amino acids composition analysis , and the result was identical to the amino acids composition of human C2peptide. The good agreement of amino acids composition , together with shown similarities of the recombinant C2peptide to C2 peptide standard in the comparative RP2HPLC analysis (Fig. 3) and IMMUL ITE C2Peptide quantitative assay , indeed suggested that the recombinant C2peptide obtained in this report was the native human C2peptide.
2. 5 Stability of recombinant proinsulin C2peptide in aqueous solution
C2peptide 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 N2terminal. Hydrolysis could take place at either the N2terminal and / or C2terminal peptide bonds adjacent to the Asp residue. The mechanism of hydrolysis undoubtedly involved intramolecular catalysis by a carboxyl group of the Asp
[ 24 ,26 ]
residue in acidic media. Ser residue could also undergo beta2elimination at alkaline conditions. In many
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cases , the beta2elimination reaction was influenced by p H and temperature. Although C2peptide could undergo degradation via a variety of chemical reactions including beta2elimination , racemization and oxidation , which were specific to certain amino acid residues , deamination of C2 terminal of polypeptide was the most common chemical pathway of polypeptide degradation under alkaline p H[ 24 - 26 ] .
With the exception of the reduced recombinant proinsulin C2peptide as peak 1 , the degradation products had not been identified in this study. The degradation products might be a form of C2peptide deamidated at the [ 27 ] end of C2terminal of C2peptide at p H 9. The C2peptide degradation showed a marked dependence on p H and temperature. Figures also suggested that C2peptide might degrade by several different specific pathways at p H 3 and p H 9 ( Fig. 4) . It was found that the p H 3 affected the degradation rate of C2peptide and that the observed degradation products increased as the degradation of C2peptide progressed and the degradation products were complicated at 70 ℃for 10 h ( Fig. 5) . While the major degradation products at p H9
appeared to be relatively stable to temperature ( Fig. 5 , Fig. 6) . No C2peptide degradation products were detected at p H 7. 4 under 37 ℃for 10 h and p H 7. 4 under 70 ℃ for 3 h while 10 g/ L BSA existed ( Fig. 5 , Fig. 6) . 99 % and 96 % of total C2peptide remained at p H 7. 4 with and without 10 g/ L BSA under 37 ℃for 10 h respectively ( Fig. 6) .
The degradation kinetics of C2peptide was also studied , Fig. 7 showed a first2order plot of the residual percentage amounts of C2peptide vs. time in various p H solution at 37 ℃or 70 ℃. It was found that p H affected the degradation first ( Fig. 4 ) . At the same p H , the temperature affected the degradation rate of C2peptide and the observed degradation reaction rates approximately followed first2order kinetics ( Fig. 7) .
Conclusively , we had successfully constructed gene carrying three copies of a C2peptide encoding gene fragments, which had been expressed in E. coli as a fusion protein at high level , about 80 mg/ L. Ni2N TA affinity chromatography efficiently separated the expressed fusion protein from the supernatant . Native C2peptide was obtained by trypsin + carboxypeptidase
Fig. 4 RP2HPLC analysis of C2peptide prepared in different pH buffers
(A) The collected C2peptide from fusion proteins enzymatic cleavage mixture before lyophilized. (B) - ( E) , The lyophilized C2peptide was prepared in different p H buffers respectively and analyzed immediately. (B) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer containing 10 g/ L BSA ; (C) p H 3 , 0. 1 mol/ L Na2titrate buffer ; (D) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer ; ( E) p H 9. 0 , 0. 1 mol/ L Tris·HCl buffer. 1 , recombinant C2peptide ; other peaks , unknown.
Fig. 5 RP2HPLC analysis of degradation of C2peptide prepared in different pH buffers kept at 70 ℃
(A) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer containing 10 g/ L BSA , 3 h ; (B) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer , 6 h ; (C) p H 3. 0 , 0. 1 mol/ L Na2titrate buffer , 10 h ; (D) p H 9. 0 , 0. 1 mol/ L Tris·HCl buffer , 10 h. 1 , recombinant C2peptide ; other peaks , unknown.
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Fig. 6 RP2HPLC analysis of degradation of C2peptide prepared in different pH buffers kept at 37 ℃for 10 h
(A) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer containing 10 g/ L BSA ; (B) p H 7. 4 , 0. 1 mol/ L Tris·HCl buffer ; (C) p H 3. 0 , 0. 1 mol/ L Na2titrate buffer ; (D) p H 9. 0 , 0. 1 mol/ L Tris·HCl buffer. 1 , recombinant C2peptide ; other peaks , unknown.
Fig. 7 First2order plot for the degradation of recombinant C2peptide in 0. 1 mol/ L Na2titrate buffer ( pH 3) and 0. 1 mol/ L Tris·HCl buffer ( pH 7. 4 or pH 9) at 37 ℃or 70 ℃
B treatment of fusion protein. The good agreement of amino acids composition , together with shown similarities of the recombinant C2peptide to C2peptide standard in the comparative RP2HPLC analysis and IMMUL ITE C2 Peptide quantitative assay , indeed suggested that the recombinant C2peptide obtained in this report was the native human C2peptide. Recombinant C2peptide was stable in p H 7. 4 buffered solution , 10 g/ L BSA showed protect effect to it in p H 7. 4 buffered solution. The activities of proinsulin C2peptide were being investigated in our laboratory.
3 4
5
6
7
Acknowledgements We thank Professor SUN Xiang2 Ming and Dr. XIA Wen2Chao for excellent assistance with RP2HPLC analysis , Dr. GU for assistance with IMMUL ITE C2Peptide quantitative assay in Nuclear Medical Laboratory of Zhongshan Hospital , and we are also grateful to Dr. ZHAN G Hui2Tang , Dr. YAN Zhi2 Qiang , Dr. CHEN Yan , Dr. SHEN Yan and Dr. YU Gu2Song for fruitful discussion and kind support .
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Kuhl C , Faber OK, Hornnes P , Jensen SL. C2peptide metabolism and the liver. Diabetes , 1978 , 27 Suppl 1 : 197 - 200
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胰岛素原 C 肽多聚体基因在大肠杆菌中的表
达 及重组 C 肽在水溶液中的稳定性
李素霞1 ,2 田丽萍2 刘海峰1 张育坚2 胡晓波2 龚 毅2 3
袁勤生1 3
( 1 华东理工大学生物反应器国家重点实验室 , 上海 200237 ; 2 中国科学院上海生命科学研究院生物工程研究中心 , 上海 200233)
摘要 合成了胰岛素原 C 肽三聚体的基因 , 并在大肠杆菌中得到高效表达 。表达的融合蛋白质通过设计的特异性位点的酶切 得
到重组的人胰岛素原 C 肽单体 。融合蛋白质以可溶性蛋白质的形式表达 , 表达量约为 80 mg/ L 。Ni2N TA 亲和柱可有效地从 细胞裂解的上清液中分离纯化融合蛋白质 , 得到纯度大于 70 %的融合蛋白质 37. 5 mg/ L 。融合蛋白质可经胰蛋白酶/ 羧肽酶 B 双酶切有效释放天然的 C 肽 。释放的 C 肽经氨基酸组成分析 、与标准对照的 RP2HPLC 分析和免疫发光法定量证实与天然人 C 肽完全相同 。C 肽经 RP2HPLC 纯化后可得 , 总的收率为 1. 5 mg/ L , 纯度大于 95 %。考察了 C 肽在冻干过程中及在水溶液中的 稳定性 。结果表明 C 肽在冻干过程中稳定 , 在水溶液中其稳定性受 p H 和温度的影响 。在 p H 3 和 p H 7. 4 缓冲液中 , 37 ℃或 70 ℃下 , C 肽的降解符合一级动力学 。C 肽冻干品直接溶解于 p H 9 的缓冲液中 , 立即降解 , 降解产物在 37 ℃和 70 ℃下基本稳定 。 C 肽冻干品直接溶解于 p H 3 的缓冲液中 , 立即发生降解反应 , 随后可观察到随温度升高和时间延长 , 降解反应的进行 , 37 ℃、
10 h , 可观察到约 80. 3 %的 C 肽保持 , 而在 70 ℃、10 h , 只有 43 % C 肽保持 。C 肽在 p H 7. 4 最稳定 , 37 ℃、6 h 或 10 g/ L BSA 存在
下 , 70 ℃、3 h , 未观察到降解产物 。PH 7. 4 、37 ℃、10 h , 在有和没有 10 g/ L BSA 存在的情况下 , C 肽的保持率分别为 99 %和 96 % , 从而显示了 BSA 的保护作用。 关键词
胰岛素原 C 肽 ; 多拷贝基因构建 ; 多拷贝 C 肽基因的表达 ; 稳定性 ; RP2HPLC
收稿日期 : 2003206224 接受日期 : 2003208218
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