Secretion expression of recombinant glucagon inEscherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

Secretion expression of recombinant glucagon inEscherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

超活性胰高血糖素的分泌表达

通过多肽化学合成方法 ,人们对胰高血糖素的结构与功能关系有了比较深刻的了解 ,其中最引人注目的成就之一是发现 [Lys17,18,Glu2 1] 胰高血糖素具有比天然胰高血糖素更高的生物活性 ,称之为超活性胰高血糖素(superactiveglucagon ,下称SA glucagon)。为了通过基因工程途径获得SA glucagon ,用PCR方法从以前构建的胰高血糖素表达载体pAGluT得到SA glucagon的基因 (SAG) ,构建了含PL 启动子 ,phoA信号肽和SAG的分泌表达载体pBLSG7。pBLSG7转化到大肠杆菌BL2 1中 ,进行SAG的分泌表达 ,在摇瓶条件下 ,该菌种能分泌表达SA glucagon达 3.6 5mg/L(A60 0 =1) ,占上清液中蛋白质的 19.5 % ,并进一步研究了诱导温度和菌株对表达的影响。     

Construction of secretory expression system suitable to express glucagon under the control of PL promoter

It was reported that PL promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His1] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD600 unit of cells in shake flask. The yield of [Des-His1] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.     

Construction of Secretory Expression System Suitable to Express Glucagon Under the Control of PL Promoter

It was reported that P_L promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His¹] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD₆₀₀ unit of cells in shake flask. The yield of [Des-His¹] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.Received: 26 September 2002 / Accepted: 24 October 2002     

Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor

The specific molecular interactions of alpha-helical peptide, human glucagon (i.e., intermolecular self-association and specific receptor-binding affinity) provided a rationale for using the glucagon as the fusion expression partner to achieve high productivity of foreign proteins both in vivo (in bacterial fusion-expression system) and in vitro (in affinity column chromatography). The fusion of glucagon peptide(s) effectively promoted homogeneous aggregate formation of recombinant proteins while avoiding intermolecular crosslinking by disulfide bridges. High sensitivity of the self-aggregation to sequence effects resulted from two distinct nonpolar domains of glucagon, determining specificity of molecular interaction and aggregate size of recombinant proteins. An N-terminal domain of glucagon molecule (Phe6-Tyr10-Tyr13) could be a certain hydrophobic moiety involved in intermolecular self-association (probably, via helix-helix docking), while a C-terminal domain (Phe22-Trp25-Leu26) seems to critically affect the oligomer size in the off-pathway aggregation of synthesized fusion proteins. An N-terminal extracellular domain of human glucagon receptor was recombinantly expressed in Escherichia coli, immobilized to a chromatography column, and efficiently renatured to a conformation that attains high specificity in interaction with N-terminus glucagon molecules of recombinant fusion proteins. Through column chromatography employing the receptor fragment as affinity ligand, the recombinant proteins were efficiently purified from total intracellular proteins, and the long-term ligand stability was evidently proven through multiple cyclic-purification experiments. Major scaffolds for using protein ligands are large-scale production in a low-cost expression system and long-term stable operation with selective-binding affinity. From this point of view, the extracellular fragment of human glucagon receptor used in this study seems to be a new potent ligand for fusion protein-based affinity chromatography.     

可溶性重组人组织因子(rhTF243)分泌型表达载体的构建和表达

目的:构建含有TF的胞外区和跨膜区基因的phoA-TF243分泌型表达载体,在大肠杆菌中以可溶形式表达重组人组织因子(rhTF243)。方法:从人胎盘组织中提取总RNA,以RT-PCR扩增获得目的基因,并克隆到phoA载体中,在大肠杆菌MM294中表达rhTF243,产物采用免疫亲合层析进行纯化。结果:通过低磷酸盐诱导工程菌,获得重组人组织因子(rhTF243) ,表达水平占菌体总蛋白量的16 .3 %,经免疫亲合层析纯化,产物纯度达到95 %以上,分子量为27 .4kD。结论:获得了rhTF243,具有与完整分子完全相同的凝血功能。     

~(21)Ala定点突变胰高血糖素及结构与功能变化

 胰高血糖素是由 2 9个氨基酸组成的多肽激素 ,具有促糖元分解的生理功能 ,其拮抗剂有治疗糖尿病病人的潜在应用价值 .在获得重组胰高血糖素基因工程菌基础上 ,利用定点突变技术改造其第 2 1位氨基酸天冬氨酸为丙氨酸 ,并经DNA测序证明胰高血糖素基因发生了点突变 .用IPTG诱导表达后 ,经亲和层析和反相高效液相层析 ,纯化到突变型重组2 1Ala 胰高血糖素 .质谱测定分子量与理论值相符 .利用园二色谱比较重组胰高血糖素和突变的2 1Ala 胰高血糖素在TFE中的二级结构 ,发现胰高血糖素以α螺旋为主要二级结构 ,2 1Ala 胰高血糖素仍有α螺旋结构特征 ,并且含量有所增大 .利用兔升血糖试验 ,发现2 1Ala 胰高血糖素生物活性比重组胰高血糖素减少 51 % (P <0 .0 1 ) .显示天然胰高血糖素第 2 1位氨基酸天冬氨酸与形成α螺旋结构关系不大 ,但在发挥胰高血糖素的生物功能中有重要作用 ,与其可作为钙离子结合位点 ,参与胰高血糖素和受体结合的潜在功能密切相关 .     

Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase.

The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+.     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

The products of Rhizobium genes, psi and pss, which affect exopolysaccharide production, are associated with the bacterial cell surface

Translational fusions between a mutant phoA (lacking its promoter, ribosomal binding site and signal peptide sequence) and Rhizobium 'symbiotic' genes were isolated. Since these fusions expressed alkaline phosphatase (AP), the product of phoA, the genes into which phoA was inserted apparently specify proteins located in the bacterial periplasm or cell membrane, the compartment in which AP has activity. These genes were psiA and genes upstream of psiA (psiA is required for normal nodule development and strains with multicopy psiA fail to make exopolysaccharide (EPS) and to nodulate). Fusions between phoA and pss (exo) genes, which are required for EPS production, also resulted in the expression of AP indicating that products of these pss genes were located at the cell surface. Using gus fusions to psiA and pssA, we found that the former was expressed in N2-fixing bean root nodules but the latter was not.     

Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Analysis of regulation of phoB expression using a phoB-cat fusion.

The phoB gene, which encodes a positive control factor for a number of phosphate-regulated genes in Escherichia coli, was cloned into multicopy plasmid pBR322. A phoB-cat fusion that expressed chloramphenicol transacetylase from the phoB promoter was constructed. Studies of the expression of the phoB-cat fusion showed that the pattern of regulation of the phoB gene was similar to that of the phoA gene, the structural gene for alkaline phosphatase. The phoB gene was derepressed under conditions of phosphate starvation, was constitutively expressed in a phoR background, and required the phoM gene product for expression in a phoR strain. Finally, a functional phoB product was required for its own synthesis. Our results indicate either that phoA gene expression responds directly to the concentration of the phoB gene product in cells or that the phoA and phoB controlling elements are quite similar.     

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.     

Generation of mice expressing the human glucagon receptor with a direct replacement vector

The process of evaluating the in vivo efficacy of non–peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non–peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of ¹²⁵I–glucagon and the ability of glucagon to stimulate cAMP production. L–168,049, a non–peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC₅₀=172±98nM) in the presence of MgCl₂ in marked contrast to the measured affinity of the murine receptor (IC₅₀=611±97nM) for this non–peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon–stimulated cyclase activity with 5 fold higher potency (IC₅₀=97.2±13.9nM) than for the murine receptor (IC₅₀=504±247nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.