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1.
T. Komai Y. Ishikawa R. Yagi H. Suzuki-Sunagawa T. Nishigaki H. Handa 《Applied microbiology and biotechnology》1997,47(3):241-245
New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control
of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into
host cells [E. coli BL-21(DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary
T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved
the infection of late-log-phase cultures of E.␣coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were
effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity
than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also
easily applicable for the expression of other proteins toxic for E. coli.
Received: 5 September 1996 / Received last revision: 1 November 1996 / Accepted: 15 November 1996 相似文献
2.
Most Escherichia coli K12 strains survive for a relatively long time outside the laboratory. Under the same conditions the isoallelic E. coli K12 relA mutants die faster because they lack the stringent response. The killing rate is increased by using a plasmid-encoded suicide system consisting of the phage T7 lysozyme gene driven by the E. coli alkaline phosphatase gene promoter (phoA). Cells containing this system were rapidly and effectively killed as soon as phosphate was made limiting. The combination of the chromosomal relA mutation and a conditional suicide system of this type provides an effective means of biological containment for recombinant E. coli strains. 相似文献
3.
Kachko A. V. Cheusova T. B. Sorokin A. V. Kazachinskaya E. I. Cheshenko I. O. Belanov E. F. Bukreev A. A. Ivanova A. V. Razumov I. A. Ryabchikova E. I. Netesov S. V. 《Molecular Biology》2001,35(3):417-422
The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the T7 RNA polymerase promoter. Recombinant NP was synthesized in Escherichia coliand in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coliand of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP. 相似文献
4.
5.
The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased
codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified
to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.
Revisions requested 13 April 2005 and 26 May 2005; Revisions received 19 May 2005 and 30 August 2005 相似文献
6.
7.
David F. Mark John W. Chase Charles C. Richardson 《Molecular & general genetics : MGG》1977,155(2):145-152
Summary The DNA polymerase induced by bacteriophage T7 is composed of a phage-specified subunit, the gene 5 protein, and a host-specified subunit, the 12,000 dalton thioredoxin of Escherichia coli. tsnC mutants of E. coli B (Chamberlin, 1974) have no detectable thioredoxin, and thus cannot support the growth of phage T7, although they are killed by phage infection. A mutant of E. coli K12 affecting thioredoxin has been isolated by a modification of the procedure used by Chamberlin (1974) to isolate tsnC mutants of E. coli B. The gene affecting thioredoxin has been designated trxA. This mutant, E. coli JM109, shows the TsnC phenotype in that it is killed by, but cannot support the growth of, bacteriophage T7. T7 DNA replication does not occur in mutantinfected cells. These phenotypic expressions of the tsnC mutation have enabled us to screen recombinants for the trxA allele in HfrxF- crosses and F-ductants in episome transfer experiments. Extracts of transductants in generalized transduction by P1 phage were screened for their ability to complement partially purified phage T7 gene 5 protein to form T7 DNA polymerase. The trxA gene is located at 84 min on the E. coli linkage map, between uvrE and metE; trxA is 34% co-transducible with metE. 相似文献
8.
Summary The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal -endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. colicells by disrupting their chromosome replicating apparatus. 相似文献
9.
Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity
of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used
inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher. 相似文献
10.
Summary Expression of the alginate lyase gene (aly) from Pseudomonas sp. W7 in Escherichia coli was very high (about 40%) and regulated by catabolite repression. The aly promoter was independent upon activation by cyclic AMP-cAMP receptor protein (cAMP-CRP) complex. When the aly promoter was removed by the construction of pKAL22, alginate lyase, which was under the control of lac promoter in E.coli, was markedly decreased (about 10%). This result confirmed that the aly promoter has stronger expression system in E.coli than lac promoter. 相似文献
11.
To improve expression ofBrevibacterium sterolicumcholesterol oxidase inEscherichia coli,we utilized theT7lacpromoter and modified the gene to encode the first 21 amino acids with high-expressionE. colicodons. These changes resulted in a 60-fold improvement of expression level. N-terminal sequencing revealed that theE. coliproduced cholesterol oxidase signal peptide is cleaved 6 amino acids closer to the N-terminus than inB. sterolicum.The recombinantE. coliproduced protein is composed of 513 amino acids with a calculatedMrof 55,374. The kinetic rate constants of the recombinant protein and theB. sterolicumproduced cholesterol oxidase are identical. 相似文献
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13.
The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs inEscherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression
of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we
adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient
reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under anE. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored
the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly,GST, which is naturally highly expressible inE. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression
cassette (which had earlier given high-level GH expression undertrc promoter) under theT7 promoter inE. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression
of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such
as the sequence context, together with the length and codons used in the first cistron are important as well. 相似文献
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15.
Gavin C. Barnard Grant E. Henderson Sriram Srinivasan Tillman U. Gerngross 《Protein expression and purification》2004,38(2):116
We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector. 相似文献
16.
A DNA sequence encodingN-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced intoEscherichia coli MM194 lysogenized with a bacteriophage having a T7 RNA polymerase gene. The amount of AAR produced by theE. coli transformant was 1100-fold more than that produced byAmycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of theE. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR andd- orl-aminoacylase.d- orl-methionine was continuously produced with a high yield fromN-acetyl-dc-methionine by the bioreactor. 相似文献
17.
The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic
sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene
under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final
desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source.
Received: 4 December 2001 / Accepted: 7 January 2002 相似文献
18.
H Takagi A Suzumura T Hoshino S Nakamori 《Journal of industrial microbiology & biotechnology》1999,23(3):214-217
The Bacillus subtilis subtilisin E gene was cloned into an expression vector of the extreme thermophile, Thermus thermophilus. Active subtilisin E was produced in E. coli, indicating that the Thermus promoter functions in E. coli. When the plasmid was further introduced into T. thermophilus, the subtilisin E gene was expressed and the gene product accumulated as an inactive pro-form, because the autoprocessing
of the wild-type enzyme to the active-form did not occur at 50°C or above.
Received 17 March 1999/ Accepted in revised form 28 June 1999 相似文献
19.
Angelika Lueking Caterina Holz Christine Gotthold Hans Lehrach Dolores Cahill 《Protein expression and purification》2000,20(3):372
We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning. 相似文献
20.
Yasukazu Nakakita Takayuki Aoyama Heiichi Sakai 《Bioscience, biotechnology, and biochemistry》2013,77(9):2385-2386
Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80°C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus. 相似文献