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1.
Al-Samarrai TH Kirk CA Jones WT Harvey D Sun X 《Protein expression and purification》2007,53(2):289-292
Recombinant Arabidopsis thaliana (At) RGL-3, using two vectors pMAL-c2 and pET 21, was expressed as inclusion bodies in Escherichia coli under a range of temperature conditions. Only low levels (8-12% of total protein) of soluble protein were produced. The "soluble" fraction was shown by native PAGE to exist as soluble aggregates of RGL-3. A method was developed, consisting of induction of expression at various temperatures that yielded high levels of refoldable inclusion bodies using the pET vector. (At) RGL-3, as inclusion bodies, was solubilized in 8M urea and refolding was initiated by 20-fold direct dilution of denaturant. Under optimal conditions, 87% of the denatured protein of inclusion bodies was successfully re-natured. Refolding was monitored by "native" PAGE. Refolded RGL-3 was shown to be present as monomers and dimers. Attempts to further purify His-tagged RGL-3 using Ni/NTA chromatography resulted in the formation of higher polymers. 相似文献
2.
Manoj Panchal 《Preparative biochemistry & biotechnology》2013,43(4):276-285
Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells. 相似文献
3.
4.
High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.
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The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein. 相似文献
5.
The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein. 相似文献
6.
Wu QY Li F Zhu WJ Wang XY 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2007,148(4):355-362
Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L− 1 of LB medium. Some parameters for the renatured and soluble forms of AK, including Km, Kd, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest kcat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques. 相似文献
7.
Naimov S Martens-Uzunova E Weemen-Hendriks M Dukiandjiev S Minkov I de Maagd RA 《Molecular biotechnology》2006,32(3):185-196
Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal
crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the
lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a
carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal
formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid
SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square
or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All
Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could
be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions.
Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude
activity against CPB, but that interfering with this crystal formation can increase the activity. 相似文献
8.
Juan José R. Coque Juan F. Martín Paloma Liras 《Molecular & general genetics : MGG》1993,236(2-3):453-458
Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a Mr of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a Mr of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus. 相似文献
9.
The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon
gamma and over-produced in the form of inactive inclusion bodies in E. coli. The amount of fusion protein was about 40% of total protein produced. The fusion protein contained in the inclusion bodies
was solubilized in 8 m urea and purified by anion-exchange chromatography. We employed the orthogonal experimental design
L16(45) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor l-arginine,
NaCl, pH, and glycine on the refolding procedure. Our results suggest that the presence of appropriate l-arginine, NaCl, and
denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein
with biological activity. The refolded fusion protein, γIFN/pk5, has in vitro anti-endothelial cell proliferation activity.
Received: 24 July 2000 / Accepted: 21 September 2000 相似文献
10.
The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins. 相似文献
11.
Wang L Zhou Q Chen H Chu Z Lu J Zhang Y Yang S 《Journal of industrial microbiology & biotechnology》2007,34(3):187-192
The gene encoding the Pyrococcus furiosus extracellular α-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant α-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved
purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90°C treatment
for 3 min in Britton–Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl
Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher
than previously reported. 相似文献
12.
An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan 总被引:1,自引:0,他引:1
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The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement. 相似文献
13.
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
14.
Giant catfish growth hormone (gcGH) cDNA was cloned and expressed in E. coli. The expected 20.5 kDa protein corresponded to the mature gcGH and was efficiently expressed. This protein was produced as inclusion bodies and comprised about 20% of total cellular proteins. The recombinant hormone promoted growth when injected intramuscularly or intraperitoneally into goldfish (Carassius auratus) at 0.1 or 1 microg soluble gcGH per g fish body wt per week. In addition, the recombinant gcGH inclusions had growth-promoting activity similar to that of the soluble form when the fish was received either by intraperitoneal injection or by oral administration. 相似文献
15.
The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed
in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under
control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially
in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of
the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically
active lipase from a basidiomycete fungus. 相似文献
16.
Cristiana Castaldo Silvia Ciambellotti Raquel de Pablo-Latorre Daniela Lalli Valentina Porcari Paola Turano 《PloS one》2013,8(8)
Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of 1H-15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease. 相似文献
17.
Juan Lin Jianhong Yao Xuanwei Zhou Xiaofen Sun Kexuan Tang 《Molecular biotechnology》2003,25(3):215-221
Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has
gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for
plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion
bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography
yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes
to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous
recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and
purification of biologically active recombinant PTA from E. coli. 相似文献
18.
Nagesh K. Tripathi Jyoti Shukla Karttik C. Biswal P. V. Lakshmana Rao 《Applied microbiology and biotechnology》2010,86(6):1795-1803
Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the
tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII)
protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies.
The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity
chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l−1 and 720 mg l−1 of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide
gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected
human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection
or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant
viral proteins. 相似文献
19.
Chimeric gene construct coding for bi-functional enzyme endowed with endoglucanase and phytase activities 总被引:1,自引:0,他引:1
Phytase and endoglucanase enzymes are being widely used as feed additives in poultry industry. In our earlier studies, the
Bacillus phytase, when expressed in Escherichia coli, was found in inclusion bodies, whereas endoglucanase was found in active soluble form. Herein, we report the development
of a chimeric gene construct coding for ~73 kDa fusion protein and its over-expression in E. coli in soluble form. The novel enzyme exhibited both endoglucanase and phytase activities across broad pH (4.0–8.0) and temperature
(25–75°C) ranges. As such, the bi-functional enzyme seems promising and might serve as a potential feed additive for enhanced
nutrition uptake in monogastric animals. 相似文献
20.
Yoshiyuki Kawaguchi Shigeo Kosugi Katsutoshi Sasaki Takeshi Uozumi Teruhiko Beppu 《Bioscience, biotechnology, and biochemistry》2013,77(7):1871-1877
The production of prochymosin directed by a cloned cDNA under the control of a trp promoter was examined in E. coli C600 and HB101. The latter host exhibited a higher degree of expression as to the production of prochymosin in the form of inclusion bodies, which accounted for more than 15 ~ 20% of the total cellular protein. The conditions for the processing of prochymosin in the inclusion bodies to active chymosin were determined. Several enzymatic properties of the processed bacterial chymosin, such as its specific activities as to milk-clotting and proteolysis, heat stability and Ca2 + dependence of the clotting activity, were almost identical to those of authentic chymosin. However, a slight difference was observed with regard to the immunological reactivity with anti-prochymosin antibody. 相似文献