Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV from Bombyx mori was synthesized according to its amino acid sequense using E .coli biased codons .The gene was cloned into the fusion expression vector pEZZ318 and was expressed in E .coli HB101.The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product .The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

在大肠杆菌体内有效地表达以组蛋白为骨架的融合蛋白HNHG

研究了在大肠杆菌体内表达了以组蛋白C 末端为骨架的融合蛋白HNHG[H(HA2 0 )N(NLS)H(组蛋白H10 的C末端 97个氨基酸 )G(GE7) ],并发现质粒DNA与其形成复合体后 ,在电镜下出现与体内染色体相同的凝聚、螺旋现象。由于HNHG的这种有效地结合质粒、并使之凝聚的特性 ,有望使之成为新兴的能将外源基因导入体内的载体系统。     

抗菌肽-X基因的克隆及在大肠杆菌中的表达

用PCR技术获得抗菌肽—X基因、TNFα基因,与温度诱导的表达载体pRC连接成为重组表达载体,导入大肠杆菌TG1,通过温度诱导表达重组蛋白。将重组质粒转入不同的表达菌中进行表达,经SDS—PAGE选出E.coil BL21(DE3)为最佳表达的宿主菌。培养后,离心得菌体,经超声破碎离心得包涵体,溶解后用CNBr切割并透析,最后经CM52纤维素柱分离纯化得到有活性高纯度的抗菌肽—X。     

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.     

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.     

Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli

Summary Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli has been achieved by the recloning of the DNA fragment encoding for larvicidal activity previously reported by us, in a pMal vector system. The potency of this recombinant strain was only 10 fold lower than the parentalB.sphaericus 1593M strain. The protein encoded was different from the previously reported larvicidal gene products ofB.sphaericus. Neverthelesss, this protein is recognized by the antiserum raised against crystal proteins. This result has indicated the presence of multiple mosquito larvicidal genes inB.sphaericus, a situation similar to that encountered withB.thuringiensis toxins.     

Expression of recombinant epidermal growth factor inE. coli

Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni²⁺-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.     

Regulated secretion and purification of recombinant antibodies inE. coli

A plasmid foroptimizedproteinexpression of recombinant Fv antibodies (pOPE) inE. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 μM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.     

Expression of cDNA fragment encoding sperm membrane peptide inE. coli

A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P _R -P _L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.     

Production of human proinsulin inE. coli in a non-fusion form

Summary A method for direct expression of the gene encoding human proinsulin inE. coli and a simple purification procedure has been established. The temperature inducible promoter is employed for rapid induction as well as high level expression, After simple down-stream processing, 80–160mg recombinant product with a purity of up to 90% can easily be obtained from 1 liter of high density fermentation medium by a single Sephadex G50 column.     

乙肝病毒前S1抗原突变体在大肠杆菌中的可溶性表达及鉴定

乙肝病毒前S1抗原含多个免疫优势表位及乙肝病毒肝细胞受体结合位点,具有重要的生物学功能。为使其高效、可溶性表达,在DnaStar软件辅助分析下,将前S1基因5′端复杂二级结构突变后,克隆入原核表达载体pQE-30a,转化大肠杆菌M15感受态细胞,经IPTG诱导后,获得了高水平、可溶性表达;并对其进行了纯化和鉴定,为进一步研究乙肝病毒前S1抗原的结构功能特点奠定了基础。     

Tinkering with transporters: Periplasmic binding protein-dependent maltose transport inE. coli

Periplasmic binding protein-dependent transport systems represent a common mechanism for nutrient and ion uptake in bacteria. As a group, these systems are related to one another and to other transporters of both prokaryotes and eukaryotes, based on sequence similarity within an ATP-binding subunit and overall structural organization. These transporters probably all use energy derived from ATP to pump substrates across membranes. Although there is considerable information about the sequences and identity of the transporters, there is little information about how they work. That is, where do ligands bind? Where do the subunits or domains interact with one another? How is the energy of nucleotide binding and/or hydrolysis converted to conformational changes? In order to address these questions we have taken a genetic approach that involves studying mutant forms of a transporter. Rather than study mutations that result in complete loss of function, the study of mutations which perturb or alter the normal function of the transporter in a defined manner has provided a limited insight into how the answers to these questions may be obtained.     

Translational control inE. coli: The case of threonyl-tRNA synthetase

Genetic studies have shown that expression of theE. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5 end of the mRNA and between 10 and 50bp upstream of the translational initiation codon in thethrS gene, is directly involved in that control. The conformation of anin vitro RNA fragment extending over thethrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constitutent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.     

Fusion expression of bovine lactoferricin in Escherichia coli

The drug resistance problem has been growing with the utilization of current antibiotics in feed and medical industries. LfcinB, a 25-amino acid antibacterial peptide derived from bovine lactoferrin, is one of potential alternatives of antibiotics. According to the bias of codon utilization of Escherichia coli, a fragment encoding LfcinB has been chemically synthesized, inserted into vector pGEX-4T-2 and expressed in E. coli. The antibacterial peptide was fused with GST with a protease cleavage site located between them. Two constructs with different cleavage sites were made. One construct, pGEX-Th-LfcinB, contains a thrombin cleavage site carried by the vector, and the other, pGEX-Th-Xa-LfcinB, contains a Factor Xa cleavage site which was introduced after the thrombin cleavage site. Fusion protein GST-Th-LfcinB protein was efficiently cleaved by thrombin, yielding recombinant LfcinB showing antibacterial activity. However, fusion protein GEX-Th-Xa-Lfcin B containing Factor Xa recognition site could not be cleaved by Factor Xa at the conditions tried in this study. Successful expression of LfcinB in E. coli provides a possible method to produce LfcinB in large amounts.     

Identification and characterization ofMycobacterium paratuberculosis recombinant proteins expressed inE. coli

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants, and it has also been isolated and identified from patients with Crohn's disease, an inflammatory bowel disease. The control of Johne's disease has been hampered by the lack of a reliable diagnostic test because of the large degree of antigenic cross-reactivity between mycobacterial and non-mycobacterial species. To help identify specific antigen(s) or epitope(s), anM. paratuberculosis expression library was screened with antibodies and DNA probes. In total, 54 clones were randomly picked, purified, and characterized by DNA probes and monoclonal antibodies with known specificity to individual mycobacterial antigens. Four clones carrying the heat shock protein 65K-, two representing the secreted protein 32K-, three representing the 21K-, and 20 clones representing the specific insertion element ofM. paratuberculosis (IS900)-encoding genes and their gene products were identified and characterized. Well-defined recombinant antigens and/or epitopes representingM. paratuberculosis may facilitate the development of specific diagnostic tests and the investigation of their role in these chronic diseases.     

Escherichia coli mutants with an altered sensitivity to cecropin D.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.     

Overexpression and purification of full-length acidic fibroblast growth factor inE.coli

Summary A full-length aFGF cDNA was cloned and inserted into a T7 expression vector for heterologous expression inE. coli. For high level production of aFGF, an optimal condition for overexpression (incubation at 42 °C for 2 hours) and a simple purification procedure employingin vitro refolding and an affinity chromatography was established. The production yield was 40–50 μg/mg of inclusion body preparation and purified aFGF showed mitogenic activity.     

Ribonuclease III reduces the efficiency of bacteriophage gy1 propagation inE. coli

TheE. coli rnc gene encodes the double-stranded, RNA-specific ribonuclease III (RNaseIII). A novel bacteriophage, gy1, was isolated, and its propagation inE. coli was shown to depend on the expression of RNaseIII in the cell. (a) gyl has a low efficiency of plating on rnc⁺ strains and a high efficiency of plating on a rnc^– E. coli strain harboring the rnc 105 point mutation that renders its RNaseIII product inactive. (b) gy1 has a high efficiency of plating on rnc^– strains in which thernc gene is disrupted by a Tn10 insertion. (c) Plasmids harboring a rnc⁺ gene that were introduced into the rnc^– strains described above reduced the efficiency of plating of gy1.