Expression of the APLA(2) Gene from Agkistrodon halys Pallas.
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The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E. coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE. The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme. The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies.Keywords:
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Summary In a systematic approach to identify genes involved in the early steps of the legume— Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid‐borne loci. A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids. Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a. New inducible loci as well as previously described genes were identified and their time course of induction determined. After initial induction, transcription of loci such as nodABC and the host‐specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids. In contrast, expression of other loci is detectable only after several hours of induction. Surprisingly, many genes remained transcribed in the nodD1 mutant suggesting the presence of other flavonoid‐dependent activators in NGR234. The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts. Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L‐fucose metabolism and its adjunction to the Nod factors.
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Objective To express the HSPC016 gene, and study the basic biological properties/characteristics of the HSPC016 gene expression products. Methods The HSPC016 gene was amplified from the plasmid of pcDNA3.1 (+)/HSPC016 by polymerase chain reaction, then subcloned into the plasmid pMD-18T and digested by two restriction enzymes of Nco Ⅰ and Hin dⅢ. After purification, the HSPC016 gene was inserted into the prokaryotic expression vector pET-28a (+), which also digested by Nco Ⅰ and Hin dⅢ. Then, the pET-28a (+) /HSPC016 was transformed into E.coli BL21 and the recombinant protein was expressed after induction by isopropyl-1-thio-β-D-galactoside. The N-terminal amino acid residual of the protein was sequenced after SDS-PAGE analysis. Results The HSPC016 gene was cloned into the pET-28a (+) successfully. The results of SDS-PAGE showed that the relative molecular weight of the expressed product was 7 200, and the expression rate was about 15%. The result of the N-terminal amino acid residual sequencing was identical to that of the molecular design. Conclusion The results suggest that HSPC016 gene is constructed successfully and expressed well in this prokaryotic expression system.
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AIM:To express tuftelin and purify it from E.coli and prepare its antiserum. METHODS :The plasmid pPRoEX TM HTc and the sequence of Tuftelin cDNA(365 amino acid)cloning in the plasmid pBluescript KS+ were cut by the restricted endonucleas.Then the two fragment were ligased by T4 DNA ligasing and the expression plasmid of fusion protein was constructed.The product was transformed into E.coli . After the induce of IPTG, the E.coli was centrifuged and breakaged by SDS loading buffer. The purified tuftelin was mixed with incomplete adjuvant, and then used to immunize rabbits. RESULTS:The product of M r 42×10 3 fusion protein was in accordance with the anticipation. 8 weeks later. the antiserum titer of 1:100 000 could be showed by the test of Western blot. CONCLUSION :The tuftelin is expressed successfully and the antiserum is prepared.
Cloning (programming)
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The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E. coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE. The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme. The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies.
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Cauliflower mosaic virus
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The recombinant plasmid pMD-eaeA was digested by the restriction enzyme BamH I and Hind Ⅲ and then the extracted eaeA gene was subcloned into prokaryotic expression plasmid pET-28a(+).After identification by PCR,restriction enzyme digestion and sequencing,the positive recombinants(designated as pET-eaeA) were transformed into E.coli BL21(DE3) pLysS.The recombinant bacteria were induced with IPTG and the expression protein was analyzed by SDS-PAGE.The result showed that a unique band was detected with the relative molecular mass of approximately 94 000,and a majority of the recombinant protein was expressed as inclusion bodies in E.coli.This protein was purified primarily and then innoculated the New Zealand rabbit three times.The antiserum was obtained and demonstrated by agar-diffusion test and Western-blotting.Therefore,the expressed fusion protein of eaeA gene of E.coli O157∶H7 had biological activity to some extent.
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Hexosaminidase
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The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.
Gene dosage
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