Expression, purification and application of bla(TEM-116) extended-spectrum beta-lactamase.
- Author:
Zhen WANG
1
;
Ying ZHENG
;
Quanshui FAN
;
Xiushu CHEN
;
Jianxin LU
Author Information
1. Institute of Microecology, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cephalosporins;
antagonists & inhibitors;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Mice;
Penicillins;
antagonists & inhibitors;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
pharmacology;
beta-Lactamases;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2010;26(2):256-263
- CountryChina
- Language:Chinese
-
Abstract:
To produce TEM-116 extended-spectrum beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-116) into expression vector pET28a(+), transforming into Escherichia coli BL21(DE3) and inducing with IPTG. We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg. The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo. Specifically, the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G (PG) when used at 10.0 IU in 1 L fermentation medium. When used at 320.0 IU, it could also degrade a mix of PG, ampicillin and cefazolin each at 200 mg in 1 L of urine. In milk, 1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG. The recombinant enzyme was fully active at the temperature ranged from 4 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(kg bw) PG in mouse models in vivo. The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.
