[Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity].
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.
Huo
Yumeng
Y
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an 271018, China.
He
Qiwei
Q
Zhao
Shuangyi
S
Xu
Yuanfang
Y
D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D010206:Papain / Q000037:antagonists & inhibitors; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; D010206:Papain