1.Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity.
Yumeng HUO ; Qiwei HE ; Shuangyi ZHAO ; Yuanfang XU
Chinese Journal of Biotechnology 2008;24(7):1194-1198
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-IdeltaD86:6His 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.
Cloning, Molecular
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Cystatins
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biosynthesis
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genetics
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Cysteine Endopeptidases
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metabolism
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Cysteine Proteinase Inhibitors
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genetics
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Escherichia coli
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genetics
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metabolism
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Genes, Plant
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genetics
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Mutation
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Oligonucleotides
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chemical synthesis
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genetics
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Oryza
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genetics
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Papain
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antagonists & inhibitors
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Prokaryotic Cells
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metabolism
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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metabolism