Cloning, expression and screening tandem repeats of the Z domain of Staphylococcus aureus protein A.
- Author:
Yi WAN
1
;
Jing ZI
;
Kun ZHANG
;
Zhimin ZHANG
;
Yuejuan ZHANG
;
Yan WANG
;
Jun WANG
Author Information
1. Molecular Biology Research Center, Shaanxi Province Microbiology Institute, Xi 'an 710043, Shaanxi, China. wanyi6565@gmail.com
- Publication Type:Journal Article
- MeSH:
Adsorption;
Bacterial Proteins;
biosynthesis;
genetics;
Chromatography, Affinity;
methods;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Immunoglobulin G;
isolation & purification;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Staphylococcal Protein A;
biosynthesis;
genetics;
Tandem Repeat Sequences
- From:
Chinese Journal of Biotechnology
2012;28(12):1500-1510
- CountryChina
- Language:Chinese
-
Abstract:
To screen an efficient recombinant Staphylococcus aureus protein A (SpA) for preparing matrix for affinity purification of immunoglobulin G (IgG), a genetic engineering approach was used to obtain monomer, two, three, four and five tandem repeats genes of the Z domain of SpA, then the genes were cloned into expression vector pET-22b and subsequently expressed in Escherichia coli BL21 (DE3). After induction with lactose, the target proteins were purified by Ni2+ affinity chromatography. The proteins with two, three, four and five tandem repeats of the Z domain were then coupled to CNBr-activated Sepharose 4B as an affinity chromatography matrix for affinity purification of human IgG. Furthermore, the differences in protein yield and IgG-binding capacity at different recombinant proteins were analyzed. The target proteins with monomer and tandem repeats of the Z domain had an effective expression in the genetic engineering bacteria. IgG could be specifically absorbed from human plasma by affinity chromatography. The protein yield and amount of IgG absorption of per mole protein could be improved by increasing the tandem repeats number of the Z domain. Compared with other tandem repeats, four tandem repeats of the Z domain exhibited more protein yield (160 mg/10 g wet cells) and higher level of IgG absorption (34.4 mg human IgG/mL gel). Therefore, four tandem repeats of the Z domain is more suitable for preparing matrix for affinity purification of IgG.
