Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity.
- Author:
Yumeng HUO
1
;
Qiwei HE
;
Shuangyi ZHAO
;
Yuanfang XU
Author Information
1. College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an 271018, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Cystatins;
biosynthesis;
genetics;
Cysteine Endopeptidases;
metabolism;
Cysteine Proteinase Inhibitors;
genetics;
Escherichia coli;
genetics;
metabolism;
Genes, Plant;
genetics;
Mutation;
Oligonucleotides;
chemical synthesis;
genetics;
Oryza;
genetics;
Papain;
antagonists & inhibitors;
Prokaryotic Cells;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2008;24(7):1194-1198
- CountryChina
- Language:Chinese
-
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-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.
