人α降钙素基因相关肽基因在酵母细胞中的分泌表达与产物的分离纯化

化学合成的人α降钙素基因相关肽(CCRP)基因用PCR法改造后使其能正确融合在酵母分泌型表达载体pVT102U/α中的α交配因子前导肽序列之后,然后进行克隆并转化酵母宿主菌S－78进行表达．培养物的上清用酶标(ELlSA)鉴定为阳性,而对照S－78、pVT102u／α为阴性,表达量用ELISA定量大于2mg／L。表达产物经阳离子交按层析(CM—Sphadex C₂₅)和HPLC纯化得到了HPLC纯产品。纯化后的CGRP能引起小鼠血压的降低,说明表达的目的蛋白既有CGRP的免疫结合活性,又有CGRP的生理活性。测定其N－端10个氨基酸序列,证明人工合成的CGRP基因在酵母细胞中已正确表达。     

Synthesis, secretion and processing of alpha-factor-interferon fusion proteins in yeast.

A gene fusion consisting of 960 base pairs of 5'-flanking region of the yeast MF alpha 1 gene, 257 base pairs coding for alpha-factor prepro sequence, and a modified human IFN-alpha 1 gene was constructed. MAT alpha cells containing the chimeric gene synthesized and secreted active IFN-alpha 1 into the growth medium. The secreted interferon molecules contained the last 4 amino acids of alpha-factor prepro sequence and the amino acids encoded by the DNA modifications introduced at the beginning of IFN-alpha 1 gene. DNA sequences coding for these amino acids were removed by oligonucleotide-directed in vitro mutagenesis. Yeast cells transformed with expression plasmids containing the altered junction synthesized and secreted human IFN-alpha 1 with the natural NH2-terminus.     

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.     

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.     

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Alpha-factor-directed synthesis of Bacillus stearothermophilus alpha-amylase in Saccharomyces cerevisiae

Promoter and leader sequence of Bacillus stearothermophilus alpha-amylase gene were removed and the gene was joined in-frame to sequences encoding the leader region of Saccharomyces cerevisiae mating pheromone alpha-factor on plasmid p69A (a hybrid of pBR322 and S. cerevisiae 2-microns plasmid). S. cerevisiae cells were transformed with plasmids containing the hybrid genes, obtaining yeast transformants which exhibit a significant extra-cellular amylolytic activity in solid medium, but not in liquid medium. Levels of alpha-amylase activity in solid medium were found to depend on the mode of fusion of the alpha-amylase gene to the alpha-factor leader region.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

Optimal conditions for the analysis of Pasteurella haemolytica lipopolysaccharide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Monomeric human calcitonin (hCT) gene and oligomeric hCT genes composed of two, three or four head-to-tail linked monomers were fused in-frame to the yeast alpha-factor leader coding sequence wild-type and fragile mutant Saccharomyces cerevisiae strains were transformed with the constructed plasmids and the yield of recombinant protein secreted into the culture medium was measured. The yeast cells secreted equal (molar) amounts of all of the hCT variants. The recombinant proteins remained stable in the growth medium for at least 3 days. The fragile cells secreted about 30% more hCT as compared to the wild-type yeast cells.     

Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via ''hybrid gene synthesis'' approach.

Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step. A 174 b.p. DNA heteroduplex, with 16 single and double base pair mismatches, was designed. One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF. The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid. After transformation in E. coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication. Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes. However, in E. coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences.     

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.     

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR

To facilitate the rapid cloning and sequencing of rearranged murine heavy-chain variable regions, we have designed a set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes. RNA was extracted from the mouse hybridoma cells secreting monoclonal antibodies: IOR-T3 (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), CB-Fib.1 (anti-human fibrin) and CB-Hep.2 (anti-hepatitis B surface antigen). First-strand cDNA was synthesized and amplified using PCR. The primers successfully amplified correct size fragments from cDNA prepared from all hybridomas. These methods will facilitate the cloning and sequencing of mouse immunoglobulin variable regions.     

Opposite and selective effects of epidermal growth factor and human platelet transforming growth factor-beta on the production of secreted proteins by murine 3T3 cells and human fibroblasts

Growth regulators such as epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) regulate the synthesis and secretion of certain proteins by cells in culture. The secretion pattern of each cell line and the effect of growth regulators on the secretion pattern are unique. EGF increased the secreted and intracellular levels of mitogen-regulated protein (MRP) and major excreted protein (MEP) by Swiss 3T3 cells. MRP is related by sequence to prolactin. MEP is a thiol protease located intracellularly in the lysosomes. EGF also selectively induced a 52,000-dalton mitogen-induced protein (MIP 52) secreted by human fibroblasts. Two types of TGF-betas were tested for their effects on the expression of secreted proteins in mouse and human fibroblasts: TGF-beta from human platelets and a growth inhibitor (GI/TGF-beta) secreted by BSC-1 cells. Each selectively decreased the levels of the two secreted proteins induced by growth factors in mouse embryo 3T3 cells and one secreted protein induced by growth factors in human fibroblasts. Platelet TGF-beta and GI/TGF-beta also induced one 48,000-dalton protein secreted by human fibroblasts. Synthesis of DNA and the incorporation of [35S]methionine into total protein in Swiss 3T3 cells were not affected by platelet TGF-beta or GI/TGF-beta. Thus, the inhibitory effect of platelet TGF-beta on the synthesis and secretion of these three proteins is due to a specific effect of platelet TGF-beta on the regulation of MRP and MEP that does not interfere with the ability of EGF to stimulate DNA or protein synthesis.     

白细胞介素-21与乙型肝炎病毒前S2S抗原融合蛋白在293T细胞中的表达

目的:构建白细胞介素-21(interleukin-21,IL-21)和乙型肝炎病毒前S2S抗原(S2S)的融合表达质粒,并研究其在293T细胞中的表达。方法:采用PCR方法扩增IL-21和HBV前S2S基因片段,分别克隆入pcDNA3真核表达质粒,用分子克隆方法构建融合表达质粒,并以脂质体2000转染293T细胞,分别应用ELISA法和Western Blot法检测细胞上清及细胞中IL-21和HBsAg的表达水平。结果:经酶切鉴定及DNA序列证实重组质粒内插入片段序列正确,三种重组质粒分别命名为pcDNA-IL-21、pcDNA-S2S和pcDNA-IL-21-S2S,并且重组质粒能在293T细胞内表达并分泌相关蛋白。结论:成功构建IL-21和乙型肝炎病毒前S2S抗原的融合表达质粒,重组质粒能在真核细胞内表达。     

Molecular cloning of mouse tumour necrosis factor cDNA and its eukaryotic expression.

Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.