卡门柏青霉-PG3脂肪酶基因的克隆、表达及活性分析

以卡门柏青霉-PG3为出发菌株,提取其总RNA,应用反转录-聚合酶链式反应(RT-PCR)扩增出860bp左右的片段,核苷酸序列分析表明其与甘油单-双酰酯脂肪酶(MDGL)基因（mdlA）一致。将此基因克隆到原核表达载体pET-28a中,转化大肠杆菌BL21(DE3),经诱导后,重组的脂肪酶（rMDGL）在宿主菌中得到表达,表达量可达菌体总蛋白量的45.2%。重组蛋白包涵体溶解、复性后用Ni²⁺组氨酸结合树脂螯合层析柱纯化,聚丙烯酰胺凝胶电泳(SDS-PAGE)显示为单一区带,其相对分子质量为41 000。以橄榄油为底物时没有检测到酶活性,以单硬脂酸甘油脂为底物时酶活性达到225nmol/s。该基因在原核系统中表达仍具有酶活性,说明MDGL的糖基化对其生物学活性并不是必不可少的。     

Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli

Recent studies in a eukaryotic system indicate that a block in secretion can lead to a block in the translation of secretory proteins. This feedback on protein synthesis is thought to be a result of an interaction of the signal recognition particle with the signal sequences of nascent proteins. Genetic studies in the prokaryote Escherichia coli suggest that a complex secretion machinery and a similar feedback mechanism exist. In addition, mutations affecting two genes, secA and secC, thought to encode components of the bacterial secretion machinery, selectively interfere with the synthesis of exported proteins. This selective interference with translation may be a result of recognition by the secretion machinery of signal sequences. If so, alteration of the signal sequence of a particular protein by mutation should eliminate the block in synthesis for that protein. We show here that signal sequence mutants for an exported protein, maltose binding protein, prevent the block in synthesis of this protein in a secA mutant.     

小鼠Pem-GST融合蛋白在大肠杆菌中的表达

目的：为制备重组小鼠Pem（以下简称mPem）-谷胱甘肽巯基转移酶（GST）融合蛋白，作为研究mPem蛋白功能的材料，方法：根据携带小鼠Pem基因编码序列的模板质粒pEGFP/mPem设计合成特异性引物，PCR扩增小鼠Pem基因编码序列，并插入融合蛋白原核表达载体pGEX-4T-3中，得到重组表达质粒pGEX-4T-3/mPem，用此重组质粒转化大肠杆菌BL21细胞，IPTG诱导重组菌表达mPem蛋白，SDS-PAGE及Western Blot鉴定表达产物。结果：重组菌株明显诱导表达出预期相对分子质量49000的融合蛋白。结论：成功构建了mPem-GST原核表达质粒，并在大肠杆菌中表达出mPem-GST融合蛋白，为mPem蛋白功能的研究打下了基础。     

牛角膜上皮45.4 kd水溶性蛋白质在脊椎动物角膜中特异性表达的免疫组化研究

用SDS 聚丙烯酰胺凝胶电泳的方法 ,制备了 45 4kd的水溶性牛角膜上皮特异性蛋白质 ,免疫家兔得到其抗体 .利用免疫组化方法研究了此蛋白质在脊椎动物角膜中表达的普遍性 .结果表明 ,此水溶性蛋白质在小白鼠、豚鼠、家兔、羊等哺乳动物和以鳖为代表的爬行动物角膜上皮细胞中呈阳性反应 ,而在鲤鱼、热带鱼、花背蟾蜍、鸡、北京雨燕等其他脊椎动物角膜中呈阴性反应     

大肠杆菌二硫键异构酶DsbA和脯氨酸异构酶PPIaseA双顺反子表达载体的构建及其在大肠杆菌中的表达

用ＰＣＲ方法获得大肠杆菌二硫键异构酶ＤｓｂＡ的编码基因ｄｓｂＡ和大肠杆菌脯氨酸异构酶ＰＰＩａｓｅＡ的编码基因ｒｏｔ,并将ＤｓｂＡ和ｒｏｔ以双顺反子形式克隆至含有Ｐｔａｃ启动子的表达载体ｐＫＫ２３３－２中。在ＩＰＴＧ的诱导下,ＤｓｂＡ和ＰＰＩａｓｅＡ获得了表达。ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：ＤｓｂＡ、ＰＰＩａｓｅＡ的表达水平分别为占菌体裂解上清液总蛋白质的４．３２％和４．０６％。     

CPC酰化酶基因的人工合成与重组表达

7-氨基头孢烷酸(7-ACA)是头孢菌素类抗生素的中间体,在医药工业中具有重要的应用价值.该文通过人工设计、合成并在重组大肠杆菌中表达头孢菌素C(CPC)酰化酶基因来实现7-ACA的一步酶法催化合成.以Pseudomonas sp.SE83菌株的CPC酰化酶蛋白质序列为模板.对CPC酰化酶基因分两段进行了全局优化设计,包括密码子替换、鸟嘌呤和胞嘧啶含量调整以及酶切位点修改等.通过装配聚合酶链式反应方法最终获得了目标基因,并克隆至表达载体pET-28a,成功构建了诱导型重组大肠杆菌E.coli BL21(DE3)/pET28-acy.经异丙基-β-D-硫代半乳糖苷诱导,在Luria-Bertani培养基中,新建重组大肠杆菌所表达的可溶CPC酰化酶占菌体总蛋白的75%以上;经优化培养基摇瓶发酵,重组菌的CPC酰化酶酶活高达2 956U/L.采用上述方法获得的CPC酰化酶,在一步酶法合成7-ACA的工业化生产中具有广阔的应用前景.     

The structure of the E. coli recA protein monomer and polymer.

The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.     

Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.