Synthesis of the Human Insulin Gene: Protein Expression,Scaling Up and Bioactivity

Abstract

Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed‐batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS‐PAGE, RP‐HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.     

人胰岛素原基因在大肠杆菌中高效外分泌表达

将胰岛素原基因融合到金色葡萄球菌蛋白Ａ的基因上,构建成大肠杆菌中基因融合的外分泌表达载体。它能高效表达且有效地分泌表达产物。利用亲和层析能方便地从培养液中分离出融合蛋白。融合蛋白经ＣＮＢｒ裂解后,经反相ＨＰＬＣ分析,分离得到具有天然结构的胰岛素原并进行了鉴定。     

人胰岛素原在甲醇酵母(Pichia pastoris)中的高效表达

甲醇营养型酵母Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ在近十几年已被人们广泛用作外源基因表达的系统。表达的是可溶性蛋白,且胞外分泌。本研究系将外源基因（胰岛素原基因）连接到穿梭质粒ＰＨＩＬ－Ｓ１上,再通过同源重组到酵母染色体,筛选表达株。用１ 升发酵罐在甲醇诱导下可获得０．３ｇ／Ｌ胰岛素原的产率。     

Renaturation of human proinsulin--a study on refolding and conversion to insulin

The production of human proinsulin in Escherichia coli usually leads to the formation of inclusion bodies. As a consequence, the recombinant protein must be isolated, refolded under suitable redox conditions, and enzymatically converted to the biologically active insulin. In this study we describe a detailed in vitro renaturation protocol for human proinsulin that includes native structure formation and the enzymatic conversion to mature insulin. We used a His(8)-Arg-proinsulin that was renatured from the completely reduced and denatured state in the presence of a cysteine/cystine redox couple. The refolding process was completed after 10-30 min and was shown to be strongly dependent on the redox potential and the pH value, but not on the temperature. Refolding yields of 60-70% could be obtained even at high concentrations of denaturant (3M guanidinium-HCl or 4M urea) and protein concentrations of 0.5mg/ml. By stepwise renaturation a concentration of about 6 mg/ml of native proinsulin was achieved. The refolded proinsulin was correctly disulfide-bonded and native and monomeric as shown by RP-HPLC, ELISA, circular dichroism, and analytical gel filtration. Treatment of the renatured proinsulin with trypsin and carboxypeptidase B yielded mature insulin.     

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line,CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.     

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.     

Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.     

人胰岛素原类似物(BKRA)基因的合成与表达

为了利用基因工程生产胰岛素,按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因,其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解,通过人工接头将２个ＢＫＲＡ基因串联起来,接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋）,实现了在大肠杆菌中的融合表达,表达产物以包含体形式存在,约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段,以ＨｉＴｒａｐ凝胶进行亲和层析,一步纯化可达纯度９５％以上．放射免疫测定表明,纯化的融合蛋白具有胰岛素抗原活性．表明已构建成人胰岛素原类似物的高效表达菌株     

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.     

从基因工程小C肽人胰岛素原类似物（B－R2－A）制备人胰岛素

以融合蛋白的形式,在Ｅ．ｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原类似物（Ｂ－Ｒ２－Ａ）．表达的融合蛋白可占细胞总蛋白６８％．经磺酸解,及初步分离Ｓ－磺酸型融合蛋白,再经ＣＮＢｒ裂解后,进行还原重组,ＨＰＬＣ分离纯化等步骤,每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５纯化,得重组人胰岛素约２０ｍｇ,其氨基酸组成与人胰岛素相同,并具有与猪胰岛素相同的生物活性．     

Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium

Leuconostoc mesenteroides dextransucrase DsrS was recombinantly produced in Bacillus megaterium and exported into the growth medium. For this purpose a plasmid-based xylose-inducible gene expression system was optimized via introduction of a multiple cloning site and an encoded optimal B. megaterium ribosome binding site. A cre mediating glucose-dependent catabolite repression was removed. Recombinant DsrS was found in the cytoplasm and exported via its native leader sequence into the growth medium. Elimination of the extracellular protease NprM increased extracellular DsrS concentrations by a factor of 4 and stabilized the recombinant protein for up to 12 h. Cultivation in a semi-defined medium resulted in a further doubling of extracellular DsrS concentration up to an activity of 65 Units/L. To develop an industrial process a high cell density cultivation of B. megaterium was established yielding cell dry weights of up to 80 g/L. After induction of dsrS expression high specific (362 Units/g) and volumetric (28,600 Units/L) activities of dextran free DsrS were measured. However, using high cell density cultivation, most DsrS was found cell-associated indicating current limitations of the production process. A protease accessibility assay identified the major limitation of DsrS production at the level of protein folding. Intracellular misfolding of DsrS hampered DsrS export via the SEC pathway at high cell densities. The subsequent use of a semi-defined mineral medium and the induction of DsrS production at lower cell densities increased protein export efficiency remarkably, but also led to extracellular DsrS aggregation. Further optimization strategies for the production of recombinant DsrS in B. megaterium are discussed.     

The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.     

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin

In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K/ILP. Pichia pastoris GS115 was transformed by pPIC9K/ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g/L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U/mg.     

Characterisation of BHK-21 cells engineered to secrete human insulin

Autoimmune destruction of cells in the pancreas leads to type I, or insulin dependent diabetes mellitus (IDDM), through the loss of endogenous insulin production capacity. This paper describes an attempt to generate artificial cells using the fibroblast cell line BHK21. Stable transfectants expressing the human preproinsulin (PPI) gene were isolated and characterised. The resulting clone selected for further analysis (BHK-PPI-C16) was capable of secreting 0.12 pmol proinsulin/hr/10⁵ cells and maintained a steady cellular proinsulin content of 0.36 ± 0.04 pmol l^–1. There was no processing of the proinsulin to mature insulin. The cells were unresponsive to glucose but there was increased proinsulin secretion in the presence of agents that stimulated formation of intracellular cAMP. Transfection of cDNAs for the key elements of the glucose sensing apparatus (GLUT2 and glucokinase) led to a subphysiological stimulation of secretion when glucokinase was transfected alone while there was a complete loss of insulin secretion when both components were overexpressed. The deleterious effect on proinsulin secretion observed upon co-expression of the glucose sensing genes may have implications for applications requiring multigene expression in BHK21 cells.     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素*

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

利用高通量生物反应器优化一种CHO细胞无血清培养基

研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。