Purification and Functional Reconstitution of the Human CHIP28 Water Channel Expressed inSaccharomyces cerevisiae

The yeastSaccharomyces cerevisiaewas used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.     

High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Saccharomyces cerevisiaewas used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducibleCUP1promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin geneSSO2, but the total level of the hormone was not improved due to impaired growth.     

Mutational properties ofsupP amber-ochre supersuppressors inSaccharomyces cerevisiae

Summary Mutational properties of thesupP amberochre supersuppressor locus inSaccharomyces cerevisiae are described. They are consistent with the proposition that thesupP locus encodes a protein.     

Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).     

Production and Purification of Recombinant Hirudin Expressed in the Methylotrophic YeastPichia pastoris

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leechHirudo medicinalis,was cloned and expressed in the methylotrophic yeastPichia pastoris.The HIR gene was inserted into theP. pastorispPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to theSaccharomyces cerevisiaepre-pro α-mating factor signal sequence. ATn903Kan^rdeterminant andHis4⁺gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into theP. pastorisstrain GS115 (his-4), His⁺transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His⁺G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a straightforward two-step chromatography procedure, the rHIR was purified to >97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data,P. pastorisoffers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.     

毕赤酵母工程菌人hepcidin的纯化及部分铁代谢活性研究

采用摇瓶培养重组毕赤酵母(Pichia pastoris)表达并分泌重组人hepcidin至胞外,经等电沉淀,凝胶过滤纯化,电泳检测样品纯度,通过Western blot检测小鼠内皮细胞中hepcidin对GFP-FPN1及TfR1表达的影响.研究发现发酵hepcidin产量达150 mg/L,纯化后经Tricine-SDS-PAGE检测为单一条带,分子质量与理论质量一致,具有抗菌活性,转染内皮细胞证实重组hepcidin可影响内皮细胞GFP-FPN1及TfR1的表达,具有调节铁代谢活性,对研究hepcidin与铁代谢的相关分子吸收机制及药物开发应用奠定了基础,对潜在的医学诊断治疗具有重要意义.     

Positive and negative regulation ofCAR1 Expression inSaccharomyces cerevisiae

Summary We localized the chromosomal targets of several of the regulatory controls of expression of theCAR1 gene. Fusion tolacZ of several fragments of the 5′ non-coding region showed that induction ofCAR1 by arginine is positively regulated by the products of theARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affectsCYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory allelesCARGRII andCARGRIII is not situated in the 5′ non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5′—nitrogen catabolite repression—activation by arginine—CARGRI-mediated repression—CAR1.     

Detection of presumptive base-pair substitution and frameshift mutations inSaccharomyces cerevisiae

Summary A rapid, economical method is described that detects presumptive base-pair substitution and frameshift mutations in the yeast,Saccharomyces cerevisiae.Research supported by the Atomic Energy Commission under contract number AT(11-1)-1314 with Illinois State University (Report number COO-1314-17).     

毕赤酵母表达重组人白细胞介素11的纯化与鉴定

报道了毕赤酵母表达人白细胞介素11的下游工艺研究,并对其产物进行了分析鉴定。所用工艺流程为离心收集上清、超滤浓缩脱盐、离子交换层析、疏水层析、凝胶过滤。所得产物经SDSPAGE电泳、RPHPLC分析、N端和C端序列分析、质谱、等电点分析和生物学活性分析,结果表明：产品纯度大于97%,结构和性质与E.coli融合表达的Neumega完全一致。     

Nitrous acid and alkylating nitrosamides: Mutation fixation inSaccharomyces cerevisiae

Summary The frequency of reverse mutants in two haploid, adenine requiring strains ofSaccharomyces cerevisiae induced with nitrous acid and alkylating nitrosamides is reduced by incubating cells after treatment at elevated temperature. Under these conditions survival is affected to a much weaker and rather variable extent. The temperature effect is independent of residual growth, and, therefore, most probably has no influenec on mutation expression.After treatment cells were plated and initially incubated at low temperature. At various intervals after plating cells were then transferred to elevated temperature. In other experiments the transfer was performed from an initial high to a final low temperature. This procedure led to the detection of a temperature sensitive phase which was reached only several hours after plating. Respreading experiments showed that the temperature sensitive phase was terminated a few hours before the number of mutant cells had doubled. It is argued that the temperature sensitive phase coincides with processes involved in the preparation of cell division, presumably DNA synthesis. Elevated temperature probably interferes with the process of mutation fixation which is assumed to consist of incorporation of altered DNA precursors or replication mistakes due to altered bases in the DNA strands.     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

重组人SNX9蛋白在大肠杆菌中的表达、纯化及性质分析

SNX9是近年发现的一种蛋白分选与转运蛋白, 属于SNX家族。将原核表达载体pET-SNX9重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达可获得分子量为70 kD的融合蛋白,Western blotting结果证明此蛋白即为目的蛋白,经检测融合蛋白主要以可溶形式表达。表达产物通过亲和层析和分子筛层析两步纯化,可以获得纯度超过95％的SNX9融合蛋白。分子筛层析中蛋白的出峰体积显示SNX9融合蛋白是以二聚体形式存在。Native-PAGE和动态光散射实验均显示其具有良好的均一性。热稳定性实验表明SNX9蛋白在15 ℃以下基本稳定。这些信息为进一步研究SNX9 的结构和功能具有重要的指导意义。     

无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测

目的:利用基因工程的方法原核表达无标签的重组人硫氧还蛋白(rhTrx)并对其进行大规模表达、纯化和鉴定.方法:从人胚胎肾HEK293细胞中提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒,重组质粒转化大肠杆菌BL21( DE3)感受态细胞,IPTG诱导表达,经两步离子交换层析纯化重组蛋白,采用SDS-PAGE、Western blotting、HPLC、MALDI-TOF-MS及经典的胰岛素二硫键还原法对重组蛋白进行鉴定.结果:构建成功了rhTrx基因表达载体;实现了rhTrx在原核细胞中的可溶性表达;纯化出的蛋白经SDS-PAGE和Western blotting分析证实为rhTrx;HPLC和MALDI-TOF-MS分析表明,纯化出的目的蛋白纯度大于95％;胰岛素二硫键还原法证实纯化出的rhTrx具有生物学活性.结论:成功构建了rhTrx的原核表达体系,建立了rhTrx的纯化和鉴定方法,为其进一步的理论研究和生产开发提供了有效基础数据.     

Expression, Purification, and Characterization of Recombinant Human Factor X

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the −2 residue (Thr → Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure–function relationships by mutational analysis.     

Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5 or 3 to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.