共查询到20条相似文献,搜索用时 31 毫秒
1.
Neophytou I Harvey R Lawrence J Marsh P Panaretou B Barlow D 《Applied microbiology and biotechnology》2007,77(2):375-381
A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous
membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying
potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion
proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused
eukaryotic protein). 相似文献
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Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
4.
Osvaldo D. Delgado M. Alejandra Martínez Carlos M. Abate Faustino Siñeriz 《Biotechnology letters》2002,24(15):1285-1290
An integrative vector was constructed to allow expression of heterologous proteins into the adhB locus of Zymomonas mobilis. As a reporter gene, the ORF of a bright variant of green fluorescent protein from Aequorea victoria (GFPuv) was fused to the adhB strong promoter from Z. mobilis by using a two-step PCR strategy. Z. mobilis recombinant strains that were stably marked by precise gene replacement at adhB locus with a single chromosomal copy of gfpuv. Protein expression was confirmed by fluorescence microscopy and measured by fluorescence spectroscopy, showing high expression levels (12 to 30 times higher than those obtained in E. coli) without affecting the host growth. 相似文献
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Veronika Altmannova Andreas Blaha Susanne Astrinidis Heidi Reichle John R. Weir 《Protein science : a publication of the Protein Society》2021,30(1):108-114
The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time‐consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N‐ and C‐terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost‐intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes. 相似文献
7.
Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides. 相似文献
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Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli. 相似文献
9.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of
PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed
a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned
into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type
PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100. 相似文献
10.
Choi SK Nishida Y Matsuda S Adachi K Kasai H Peng X Komemushi S Miki W Misawa N 《Marine biotechnology (New York, N.Y.)》2005,7(5):515-522
A complementation analysis was performed in Escherichia coli to evaluate the efficiency of β-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25ΔcrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the
E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion
by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic
positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into α-Proteobacteria. 相似文献
11.
Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955 总被引:11,自引:0,他引:11
Yves Dessaux Jacques Tempé Stephen K. Farrand 《Molecular & general genetics : MGG》1987,208(1-2):301-308
Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli. 相似文献
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Muhammad S. Nadeem Mohammed Razeeth Hani M. Z. Choudhry Firoz Anwar Mazin A. Zamzami Bibi N. Murtaza Fahad A. M. Al-Abbasi Mohammad I. Khan Abdul R. Shakoori 《Journal of cellular biochemistry》2020,121(1):125-134
Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules. 相似文献
13.
Host cell physiology is known to play a crucial role in the expression of foreign genes in heterologous systems. Expression
of archaeal genes in anaerobic or semi-anaerobic growth conditions of E. coli has been previously reported to be a means of improving solubility of some proteins. Here, we report that some of the Rosetta
strains of E. coli, which harbour the rare tRNA genes for the expression of archaeal genes, favour semi-anaerobic conditions for the expression
of putative FMN binding domain of glutamate synthase from Methanocaldococcus jannaschii at low inducer concentrations. 相似文献
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【目的】利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。【方法】从大肠杆菌BAP1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npg A表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。【结果】利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。【结论】在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。 相似文献
16.
Seleem M Ali M Al-Azeem MW Boyle SM Sriranganathan N 《Applied microbiology and biotechnology》2007,75(6):1385-1392
The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein
is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a
single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover,
the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has
the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification. 相似文献
17.
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
18.
Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp. N1. The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp. N1. It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom. 相似文献
19.
Horton CE Huang KX Bennett GN Rudolph FB 《Journal of industrial microbiology & biotechnology》2003,30(7):427-432
Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and
ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum.
ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production. 相似文献
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Lena Anton Katariina Majander Harri Savilahti Liisa Laakkonen Benita Westerlund-Wikström 《Microbial cell factories》2010,9(1):97