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Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity / 生物工程学报
Chinese Journal of Biotechnology ; (12): 1194-1198, 2008.
Article in Chinese | WPRIM | ID: wpr-275404
ABSTRACT
According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD866His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Oligonucleotides / Prokaryotic Cells / Oryza / Recombinant Fusion Proteins / Cysteine Endopeptidases / Papain / Cystatins / Cysteine Proteinase Inhibitors / Cloning, Molecular / Genes, Plant Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2008 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Oligonucleotides / Prokaryotic Cells / Oryza / Recombinant Fusion Proteins / Cysteine Endopeptidases / Papain / Cystatins / Cysteine Proteinase Inhibitors / Cloning, Molecular / Genes, Plant Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2008 Type: Article