共查询到20条相似文献,搜索用时 31 毫秒
1.
Charles Van der Henst Caroline Charlier Michaël Deghelt Johan Wouters Jean-Yves Matroule Jean-Jacques Letesson Xavier De Bolle 《BMC microbiology》2010,10(1):248
Background
When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active. 相似文献2.
The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMB 9871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111 units g–1 cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone. 相似文献
3.
M. V. Padkina L. V. Parfenova A. E. Gradoboeva E. V. Sambuk 《Applied Biochemistry and Microbiology》2010,46(4):409-414
The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed.
There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed
that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested
that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features
of its metabolism. 相似文献
4.
Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL
(R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion
bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography
was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then
subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully
cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein. 相似文献
5.
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
6.
Doris Hartinger Stefan Heinl Heidi Elisabeth Schwartz Reingard Grabherr Gerd Schatzmayr Dietmar Haltrich Wulf-Dieter Moll 《Microbial cell factories》2010,9(1):62
Background
Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. 相似文献7.
Santosh Kumar Sahu Archita Rajasekharan Sathyanarayana N. Gummadi 《Biotechnology letters》2009,31(11):1745-1752
Human phospholipid scramblase 1(hPLSCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents
and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 DE3). Addition of 3% (v/v) ethanol before induction and decreasing the post-induction temperature to 15°C increased
the solubility of hPLSCR1 to ~10 and ~15% respectively. Presence of groES-groEL complex solubilized the hPLSCR1 to ~30% of the total hPLSCR1. Absence of groES-groEL did not improve the solubility of hPLSCR1 suggesting that groES and groEL are the essential chaperones for the correct folding of hPLSCR1 when over-expressed in E. coli. 相似文献
8.
Withu Choosri Regina Paukner Petra Wührer Dietmar Haltrich Christian Leitner 《World journal of microbiology & biotechnology》2011,27(6):1349-1353
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength
LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg
of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased
the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity
chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had
kinetic properties similar to those of the fungal wild-type enzyme. 相似文献
9.
Bo Hee Maeng Dong Hyun Nam Yong Hwan Kim 《World journal of microbiology & biotechnology》2011,27(6):1391-1398
Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides.
In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic
monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this
study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP
in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets
included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these
chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most
efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of
the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed
without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of l-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity
was 3.5 times higher compared with that of no induction with l-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration
(1.0 mM l-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of
anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold
improperly folded anti-BNP scFv. 相似文献
10.
In the yeast Saccharomyces cerevisiae, the molecular chaperone HSP26 has the remarkable ability to sense increases in temperature directly and can switch from
an inactive to a chaperone-active state. In this report, we analyzed the effect of expression of HSP26 in Arabidopsis thaliana plants and their response to high temperature stress. The hsp26 transgenic plants exhibited stronger growth than wild type plants at 45 °C for 16 h. The chlorophyll content and chlorophyll
fluorescence decreased much more in wild type than in transgenic plants. Moreover, the transgenic plants had higher proline
and soluble sugar contents, and lower relative electrical conductivity and malondialdehyde contents after high temperature
stress. Furthermore, we found that over-expression of HSP26 in Arabidopsis increased the amount of free proline, elevated the expression of proline biosynthetic pathway genes and therefore enhanced
Arabidopsis tolerance to heat stress. 相似文献
11.
Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic
expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase
and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic
activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant
strain was induced by 0.7 mmol l−1 isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic
of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases. 相似文献
12.
van der Heide M Hollenberg CP van der Klei IJ Veenhuis M 《Applied microbiology and biotechnology》2002,58(4):487-494
We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomvces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P(MOX)) on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P(MOX) induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains. 相似文献
13.
Rosa Garcia Sanchez Bärbel Hahn-Hägerdal Marie F Gorwa-Grauslund 《Microbial cell factories》2010,9(1):40
Background
In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. 相似文献14.
Komeda T Yurimoto H Kato N Sakai Y Kondo K 《Molecular genetics and genomics : MGG》2003,270(3):273-280
The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5 non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.Communicated by C. P. Hollenberg 相似文献
15.
Ning-Ning Song Yan Zheng Shi-Jin E Duo-Chuan Li 《Journal of microbiology (Seoul, Korea)》2009,47(1):123-130
A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids
extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and
Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN3, but not by KCN or H2O2. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved
in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The
protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg.
The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at
50 and 60°C and the half-life at 80°C was approximately 40 min. 相似文献
16.
Chenguang Zhu Zhengkai Xu Rentao Song 《World journal of microbiology & biotechnology》2011,27(12):2863-2871
A Bacillus subtilis strain BEC-1 demonstrating high carboxymethylcellulose-degrading activity was isolated from the forest soil sample. In order
to characterize the biochemical specialty of its cellulase, the endoglucanase gene egl173 was cloned from this strain and was expressed in Escherichia coli. The gene encoded a protein of 499 amino acids with a molecular weight of 64 kDa. The purified Egl173 could hydrolyze both
soluble and insoluble celluloses with distinct activities. This enzyme showed the highest enzyme activity at pH 4, maintained
at least 85% activity in the pH range of 3–7, displayed maximum activity at 60°C and was highly stable between 30 and 60°C.
It was found that this endoglucanase was increasedly active and retained its high stability after incubation with 5 M NaCl
or 3 M KCl for 24 h. Furthermore, after incubation with 10 mM of dithiothreitol, the enzyme activity was significantly enhanced
(125% of the control level). In the presence of diverse metal ions (except mercury and manganese cations), organic solvents,
surfactants (except SDS) and chelating agent, this enzyme kept more than 85% active. This halo-tolerant, acidophilic and highly
stable endoglucanase is prospectively to be exploited as the advanced enzymatic product for diverse industrial applications. 相似文献
17.
Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm−3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T0 putative transformants and their seed progenies (T1 to T3 generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T0 – T3 plants were morphologically normal and fertile.This research was supported by the National Agency for Agricultural Research (grants No. QE 0046 and QF 3072) and Ministry of Education of the Czech Republic (grant No. ME 433). 相似文献
18.
Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids
into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study,
Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared
with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al
tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content
and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments
showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the
transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression
of mitochondrial CS can be a useful tool to achieve Al tolerance. 相似文献
19.
20.
l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from
E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers
formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented. 相似文献