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We describe an expression vector that functions in enteric bacteria. The vector contains the coliphage λ promoters PR and PL and entire PR and PL operators in tandem upstream from the multiple cloning sites containing the kanamycin-resistant gene. The vector also specifies a ribosome binding site and a thermolabile repressor, cI857, and the PRM promoter. These promoters as well as lacUV5 and trp promoters were inserted into the EcoRI site of pKO-1 plasmid so that they drove the expression of a reporter gene, galactokinase (galK). The PRPL promoter showed the highest efficiency of galK expression in the Escherichia coli strain K12ΔH1Δtrp; it was strong in Klebsiella aerogenes, and weak in Serratia marcescens and Citrobacter freundii. 相似文献
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G. -P. Xue J. S. Johnson D. J. Smyth L. M. Dierens X. Wang G. D. Simpson K. S. Gobius J. H. Aylward 《Applied microbiology and biotechnology》1996,45(1-2):120-126
Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities
(units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher
when compared with cells grown in the presence of the inducer isopropyl β-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacI
q, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λPL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λPL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated
Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated
that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.
Received: 27 June 1995/Received revision: 13 September 1995/Accepted: 30 September 1995 相似文献
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Expression of single-chain antibody fragments (scAb)in the periplasm ofEscherichia colioften results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing theE. coliSkp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl β-
-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes. 相似文献
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Employment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB2 ABC Transporter in Escherichia coli 总被引:1,自引:0,他引:1
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Christopher D. Rice Jacob E. Pollard Zachery T. Lewis William R. McCleary 《Applied microbiology》2009,75(3):573-582
Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (Pi). Under these conditions, the high-affinity PstSCAB2 protein (i.e., with two PstB proteins) is the primary Pi transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the Ptac promoter and the lacOID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized PphoB::Ptac and PpstS::Ptac strains to characterize phenotypes resulting from various ΔphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB2 transporter, as well as its abundance within the cell. In addition, we used the PphoB::Ptac ΔphoU strain as a platform to begin characterizing new phoU mutations in plasmids. 相似文献
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Phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2, is a phospholipid which plays important roles in clathrin-mediated endocytosis. To investigate the possible role of this lipid on viral entry, two viruses important for animal health were selected: the enveloped vesicular stomatitis virus (VSV) − which uses a well characterized clathrin mediated endocytic route − and two different variants of the non-enveloped foot-and-mouth disease virus (FMDV) with distinct receptor specificities. The expression of a dominant negative dynamin, a PI(4,5)P2 effector protein, inhibited the internalization and infection of VSV and both FMDV isolates. Depletion of PI(4,5)P2 from plasma membrane using ionomycin or an inducible system, and inhibition of its de novo synthesis with 1-butanol revealed that VSV as well as FMDV C-S8c1, which uses integrins as receptor, displayed a high dependence on PI(4,5)P2 for internalization. Expression of a kinase dead mutant (KD) of phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K-Iα), an enzyme responsible for PI(4,5)P2 synthesis that regulates clathrin-dependent endocytosis, also impaired entry and infection of VSV and FMDV C-S8c1. Interestingly FMDV MARLS variant that uses receptors other than integrins for cell entry was less sensitive to PI(4,5)P2 depletion, and was not inhibited by the expression of the KD PIP5K-Iα mutant suggesting the involvement of endocytic routes other than the clathrin-mediated on its entry. These results highlight the role of PI(4,5)P2 and PIP5K-Iα on clathrin-mediated viral entry. 相似文献
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Fang Yang Quan-Quan Ji Liang-Liang Ruan Qing Ye En-Duo Wang 《The Journal of biological chemistry》2014,289(30):20953-20959
There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (ΔNhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the ΔNhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells. 相似文献
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Silvia C. Locatelli-Hoops Inna Gorshkova Klaus Gawrisch Alexei A. Yeliseev 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2045-2056
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2. 相似文献
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Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase 总被引:1,自引:1,他引:0
Andrew Darwin Haitham Hussain Lesley Griffiths Jane Grove Yoshihiro Sambongi Stephen Busby Jeff Cole 《Molecular microbiology》1993,9(6):1255-1265
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PII signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, PII binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, PII (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of PII binding to NAGK, we searched for PII variants with altered binding characteristics and found PII variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type PII protein. Analysis of interactions between these PII variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the PII I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the PII I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of PII proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus PII after having latched onto NAGK. Moreover, both PII I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of PII-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of PII initiates the bending of the extended T-loop of PII, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. 相似文献
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The ability of aflatoxins B1 and G1 to induce back mutations to arg+ in Escherichia coli K-12/343/113 was compared with induction of mitotic gene conversion to ade+ in the diploid yeast strain Saccharomyces cerevisiae D4, ade2?. In analogy to previous results with other microorganisms, the compounds were not genetically active per se, indicating that under the experimental conditions employed none of the tester strains were able to activate the compounds to mutagenic products.In experiments using liver homegenates (S-9 fraction) of male Golden Syrian hamsters previously treated with phenobarbital, aflatoxin B1 exhibited strong genetic activity both in E. coli and in S. cerevisiae, whereas the mutagenic activity of aflatoxin G1 was markedly lower and could be detected only in the E. coli tester strain. These results correlate the findings that aflatoxin G1 is a less potent carcinogen and mutagen than aflatoxin B1. 相似文献
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Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa 总被引:24,自引:11,他引:13
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Michael L. Vasil Randy M. Berka Gregory L. Gray Hiroshi Nakai 《Journal of bacteriology》1982,152(1):431-440
Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (Pi)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by Pi as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described Pi-repressible proteins of P. aeruginosa. The existence of a Pi regulon of P. aeruginosa is proposed. It is likely that one of these genes also regulates the level of pyocyanin production by P. aeruginosa and that one or more play a role in transport or binding of Pi. The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence of P. aeruginosa and in the study of the genetics and physiology of Pi-regulated proteins. 相似文献
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《Journal of biotechnology》1987,6(1):71-81
The expression of several genes in Escherichia coli under the control of the lambda pR promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration. 相似文献
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Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase
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Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable PBAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a PBAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield. 相似文献