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1.
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. 相似文献
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A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. 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. 相似文献
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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。 相似文献
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Antibacterial peptides have received increasing attention as a new pharmaceutical substance. But the molecular mechanism of lysis is still poorly understood. CMIV gene and mutant CMIV gene in GST fusion system were expressed. After cleaving with different cleavage reagents, the peptide with an excess of N-terminus and with an un-amidated C-terminus stopped the activity while the peptide with an excess Asn at the C-terminus had the activity level the same as natural CMIV. The results showed that the terminal structure of cecropin CMIV played an important role in its biological activity. 相似文献
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Kwang Soo Ron Mo Sun Kwon Young Hwan Do Seung Dal Song Hwa Sook Chung Jong Suk Song 《Journal of Plant Biology》1998,41(3):233-239
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. 相似文献
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Eun-Jung Shin So-Lim Park Sung-Jong Jeon Jin-Woo Lee Young-Tae Kim Yeon-Hee Kim Soo-Wan Nam 《Biotechnology and Bioprocess Engineering》2006,11(5):414-419
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. 相似文献
10.
The terminal structure plays an important role in the biological activity of cecropin CMIV 总被引:1,自引:0,他引:1
Antibacterial peptides have received increasing attention as a new pharmaceutical substance. But the molecular mechanism of lysis is still poorly understood. CMIV gene and mutant CMIV gene in GST fusion system were expressed. After cleaving with different cleavage reagents, the peptide with an excess of N-terminus and with an un-amidated C-terminus stopped the activity while the peptide with an excess Asn at the C-terminus had the activity level the same as natural CMIV. The results showed that the terminal structure of cecropin CMIV played an important role in its biological activity. 相似文献
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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. 相似文献
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Chang Shin Yoon Eun Gyu Lee Young Seek Lee Il Yup Chung 《Biotechnology and Bioprocess Engineering》1997,2(2):86-89
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. 相似文献
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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. 相似文献
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Yuehau Li Yuan Chang Yan Wei Guo Feng Zhao Jun Lai S. S. Koide 《Molecular biology reports》1995,21(2):105-112
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. 相似文献
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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. 相似文献
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Mathias Springer Monique Graffe Jacques Dondon Marianne Grunberg-Manago Pascale Romby Bernard Ehresmann Chantal Ehresmann Jean-Pierre Ebel 《Bioscience reports》1988,8(6):619-632
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. 相似文献
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Howard A. Shuman Cynthia H. Panagiotidis 《Journal of bioenergetics and biomembranes》1993,25(6):613-620
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. 相似文献
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El-Zaatari Fouad A. K. Naser Saleh A. Engstrand Lars Hachem Charles Y. Graham David Y. 《Current microbiology》1994,29(3):177-184
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. 相似文献
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应用多聚酶链反应(PCR),直接从SIV感染的猴艾滋病(SAIDS)模型猴的外周血淋巴细胞总DNA中扩增出767bp的SIV核心蛋白P27基因片段。扩增产物经EcoRI及SalI双酶切后,克隆入相同酶切的表达质粒pBV220中,获得含SIV核心蛋白基因片段的重组质粒pBVSG,并进行DNA序列分析。用该重组质粒转化大肠杆菌DH5a经筛选、增殖及42℃温度诱导,SDS-PAGE表明外源基因表达蛋白含量占菌体总蛋白14.5%,Western-blot证实表达产物能被SIVP27单克隆抗体及SAIDS模型猴血清中特异性抗体识别。 相似文献