E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide

The recA protein mediates both genetic recombination and several cellular responses to DNA damage, including the induction of temperate bacteriophage. Indication of phage lambda results from proteolytic cleavage of lambda repressor directed by recA protein. We show here that this cleavage reaction requires both polynucleotide and ATP. We suggest that a stoichiometric complex of recA protein and DNA is active both to destroy repressors by proteolytic cleavage and to initiate pairing of this DNA to its homologous sequence in a DNA duplex ('strand invasion').     

The E. coli ffh gene is necessary for viability and efficient protein export.

Homologues of the gene encoding the 54K (M(r) 54,000) subunit of the mammalian signal recognition particle have been identified in different organisms. The Escherichia coli homologue, termed ffh (for fifty-four homologue), specifies a protein (Ffh) that shares many properties with its eukaryotic counterpart, including association with mammalian 7S RNA and the ability to bind signal sequences specifically. Ffh also associates with E. coli 4.5S RNA, showing that it can form a ribonucleoprotein complex in prokaryotes. These results are intriguing because extensive genetic and biochemical characterization of E. coli failed to identify a signal recognition particle-like mechanism for protein export. Here we address this issue directly by construction of a strain in which ffh expression is arabinose-dependent. Results of depletion experiments indicate that Ffh is important in protein translocation.     

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％。     

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。     

在耐药性研究中的大肠杆菌

随着抗生素应用于临床和生产,许多疾病得到了较好的控制,同时也出现了细菌的耐药问题.大肠杆菌能够通过畜禽产品的加工及储藏等传播给人类,许多耐药菌株引起的疾病治疗非常困难.本文就大肠杆菌耐药性的研究现状、耐药原因、耐药机制、以及耐药性的消除做一扼要概述.     

Identification and characterization of E. coli type-1 pilus tip adhesion protein

The type-1 pilus of Escherichia coli is the prototype of this class of hair-like, multimeric adhesive organelles. This pilus mediates adherence to mannose-containing receptors on mucosal epithelia and other cells. The type-1 pilus, in one of several serological variants, is expressed by nearly all E. coli strains, and its promotion of colonization by pathogenic bacteria and the protective effects of purified pilus vaccines suggest that it is important as a bacterial virulence factor. Both the adhesive function and the serological variation of the type-1 pilus have been attributed to the thousand or so pilin protein monomers making up the pilus rods. This idea has been contradicted by our earlier observations on an E. coli strain expressing adhesion-defective pili. More recent genetic evidence also indicates that auxiliary pilus proteins are required for adhesive function. We report here the identification of three previously undetected integral minor proteins on the type-1 pilus, and show that one of them is the receptor-binding adhesin. This protein is antigenically conserved among strains with different pilin serotypes and is located at the pilus tip.     

Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli

Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3A reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore. Putative ganglioside GM1-binding sites on the B subunits are more than 20A removed from the membrane-crossing A1 subunit. This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin.     

人表皮生长因子融合蛋白在大肠杆菌表达的研究

在大肠杆菌中高效表达人表皮生长因子(hEGF)。根据大肠杆菌对密码的偏爱性,构建高效表达EGF融合蛋白的菌株,经离子交换层析和凝胶过滤层析两个步骤使产物得到纯化。融合蛋白表达产量为27%,EGF融合蛋白纯化产物纯度为95%以上,促3T3细胞增殖的活性测定为ED50为4.2ng/mL。在pET 3c:BL21(DE3)大肠杆菌表达系统中,以融合蛋白方式表达人表皮生长因子,表达效率高,纯化路线简单,产量较高,适合产业化生产。     

Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E. coli

When present in single-stranded DNA, palindromic or quasi-palindromic sequences have the potential to form complex secondary structures, including hairpins, which may facilitate interstrand misalignment of direct repeats and be responsible for diverse types of replication-based mutations, including deletions, additions, frameshifts and duplications. In regions of palindromic symmetry, specific deletion events may involve the formation of a hairpin or other DNA secondary structures which can stabilize the misalignment of direct repeats. One model suggests that these deletions occur during DNA replication by slippage of the template strand and misalignment with the progeny strand. The concurrent DNA replication model, involving an asymmetric dimeric DNA polymerase III complex which replicates the leading and lagging strands, has significant implications for mutagenesis. The intermittent looping of the lagging strand template, and the fact that the lagging strand template may contain a region of single-stranded DNA the length of an Okazaki fragment, provides an opportunity for DNA secondary-structure formation and misalignment. Here we report our design of a palindromic fragment to create an 'asymmetric palindromic insert' in the chloramphenicol acetyltransferase gene of plasmid pBR325. The frequency with which the insert was deleted in Escherichia coli depends on the orientation of the gene in the plasmid. Our results suggest that replication-dependent deletion between direct repeats may occur preferentially in the lagging strand.