猪肥胖基因在大肠杆菌中的高效融合表达

采用PCR方法扩增猪肥胖基因编码原成熟蛋白cDNA序列,并在5′端加上BamHⅠ位点,3′端加上EoRⅠ位点,将5′端密码子CCC转变为大肠杆菌常见密码子CCG,扩增得到459bp的片段,克隆于融合表达载体p GEX-2TBamHⅠ和EcoRⅠ位点,酶切、测序正确,经0．1mmol/LIPTG诱导表达出一条约42kD的融合蛋白,其中26kD为pGEX-2T中带有的谷胱苷肽转移酶,16kD是猪肥胖基因表达产物瘦蛋白。利用非融合表达产品制备抗血清,检测融合表达的重组蛋白,Western-blot为阳性。     

外源基因在叶绿体表达系统中高效表达研究进展

综述叶绿体表达系统的特点,转化方法,同质化研究及提高外源基因在叶绿体基因组中表达水平的研究状况。     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置：胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和途径。     

外源基因在大肠杆菌中表达研究进展

近年来,基因工程技术的迅速发展,大量有价值的蛋白质大肠杆菌中获得了高表达。多种表达系统的完善与发展,及蛋白质分离纯化技术的提高,异源蛋白的产量与纯度已不再是困扰人们的主要问题,人们开始更多地关注异源蛋白的活性,比活性及异源蛋白的正确性,完整性。随着这些问题的解决,重组蛋白的应用才能真正走向成熟。     

优化外源基因在大肠杆菌中表达的翻译起始率的策略 …

ｍＲＮＡ的翻译起始区（ＴＩＲ）的二级结构对翻译起始率有很大的影响。本文建立了一种改进外源基因在大肠杆菌中翻译起始率的系统。以人分裂细胞核抗原（ＰＣＮＡ）基因为模型,将ＰＣＮＡ基因５＇端编码区的１１４ｂｐ的顺序插入质粒ｐＴＺ１９Ｒ中ＬａｃＺ＇的５＇端构成融合基因。用定点突变法在ＰＣＮＡ的ＡＵＧ的－８位插入一个Ｓｈｉｎｅ／Ｄａｌｇａｒｎｏ（ＳＤ）顺序ＧＡＧＧＴ,再以合成的带部分随机序列寡核苷酸作引物,     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置:胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和用途 。     

外源基因在转基因植物中的表达

外源基因在转基因植物中的表达王忠华夏英武舒庆尧（浙江农业大学核农所,杭州３１００２９）近十几年来,人们通过各种方法将外源基因导入植物体内产生许多转基因植物,包括水稻、小麦、棉花、烟草、大豆、番茄、马铃薯等重要粮食作物和经济作物。据不完全统计目前已获得...     

高温乳糖酶基因在大肠杆菌中的高效表达

来源于嗜热脂肪芽孢杆菌（Bacillus stearothermophilus)的β-半乳糖苷酶基因bgaB经克隆,测序后,转入大肠杆菌高效表达载体pET-20(b)中,重组菌在IPTG诱导下,表达出的重组蛋白比酶活量为6.66U/mg。比出发菌株高50倍。     

大肠杆菌中外源基因的表达调节

大肠杆菌已经被广泛地应用于表达各种外源基因,但基因的表达受到多种因素的调节,而且不同的外源基因在大肠杆菌中的表达效率也有很大差异。本文从转录水平调节、翻译水平调节、培养条件调节等方面综述了大肠杆菌中外源基因的表达调节,以便认识其规律,有助于使用有效的方法提高外源基因在大肠杆菌中的表达效率。     

Lactobacillus casei磷脂酶A_2基因在大肠杆菌中的重组表达

以Lactobacillus casei染色体基因组为模板,PCR扩增获得磷脂酶A2基因pla2,以pET-28a(+)为载体构建重组表达质粒pET-28a(+)-pla2。通过IPTG诱导实现磷脂酶A2在E.coli DE3中的重组表达。对诱导条件初步优化后,重组菌酶活最大可达2.8 U/mL。通过Ni-螯合柱对目的蛋白进行纯化,SDS-PAGE分析重组磷脂酶A2相对分子质量为1.7×104。通过酶学性质分析,最适温度为37℃,最适pH 8,比酶活为110 U/mg。     

Improved glutathione production by gene expression in Escherichia coli

AIMS: To improve glutathione (GSH) production in Escherichia coli by different genetic constructions containing GSH genes. METHODS AND RESULTS: GSH production was very low in E. coli by the expression of gshI gene. An increase of GSH production was achieved by the expression of both gshI and gshII genes in E. coli. A higher GSH production, namely 34.8 mg g(-1) wet cell weight, was obtained by simultaneous expression of two copies of gshI gene and one copy of gshII gene. CONCLUSIONS: The simultaneous expression of two copies of gshI gene and one copy of gshII gene resulted in a significant increase in GSH production. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression strategy for GSH production described here can be used to increase gene expression and obtain high production rates in other multienzyme reaction systems.     

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18

In this study, Agilent two‐colour microarray‐based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full‐sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two‐fold change minimum threshold, we found 18 genes that were differentially expressed (10 up‐regulated and eight down‐regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation‐ and immune‐related pathways. Based on the genes identified in the screen and the pathway analysis results, real‐time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid‐related pathways), SLA‐1 and SLA‐3 genes (of inflammation‐ and immune‐related pathways), as well as the differential genes FUT1, TAP1 and SLA‐DQA. Subsequently, real‐time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.     

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Expression of the Streptomyces griseusα-amylase gene in Escherichia coli

Abstract The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular α-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no α-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

HER2胞外区基因的克隆及其在大肠杆菌中的可溶性表达

采用反转录PCR和PCR方法分别克隆P185^HER2/neu胞外区基因和噬菌体M13K07g3p—N1结构域基因,然后将二偶联入pET-22b( )载体中,在大肠杆菌中进行融合表达。可溶性目的蛋白表达量占细菌可溶性表达产物总量的30％72右．并通过镍亲和层析纯化出目的蛋白。以上结果为从噬菌体抗体库中筛选抗P185^HER2/neu的抗体奠定了基础。     

D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达

以D-乳酸高产菌菊糖芽胞乳杆菌Y2-8基因组DNA为模板,通过PCR扩增得到960 bp的磷酸果糖激酶基因(pfk)。氨基酸序列比对分析表明,该磷酸果糖激酶(PFK)与其他乳酸菌PFK具有保守的底物结合位点,但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上,获得重组菌E-pSE-pfk。进一步通过诱导条件的优化,重组菌的PFK比酶活达到4.89 U/mg,是优化前的4.79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。     

Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production

In this study, the expression level of the pyc gene from Lactococcus lactis was fine tuned to improve succinate production in Escherichia coli SBS550MG. IPTG induction in the cultures of SBS550MG with pHL413, a positive control plasmid previously constructed (Sanchez et al., 2005), gave drastically decreased PYC activity and succinate yield. We constructed several plasmids for the expression of pyc to change copy number and variant promoters. Among the constructs, as compared to pHL413, the PYC activity dropped significantly with the Plac, Ptac, Ptrc or native Ppyc promoters in medium or high copy vectors, which resulted in a decrease in succinate yield. Three constructs pThio12, pHL413-Km, and pHL413-Km(lacIq-)N showed considerable PYC activity and improved succinate production in E. coli SBS550MG. The native Ppyc promoter was also modified in order to vary pyc expression levels by site-directed mutagenesis of the −10, −35, −44 regions, and the spacer regions between −10 to −35 and −35 to −44 regions. Out of 9 native promoter variants, the MIII variant resulted in a 20% increase in PYC activity, and improved succinate yield in SBS550MG. We also determined the copy number and stability of pHL413 and pHL413-Km. The two plasmids showed roughly the same copy number, but the pHL413-Km plasmid was relatively more stable. This study provides more understanding of the plasmid characteristics and fine tuning of the expression level of pyc for optimization of the succinate production processes.