Prokaryotic expression, purification and enzymatic properties of nuclease P1.
- Author:
Yanan WANG
1
;
Aiyun WEI
;
Meiyan WANG
;
Xiaobin WEI
;
Chao ZHANG
;
Liwei SHAN
;
Sanhong FAN
Author Information
1. College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Enzyme Stability;
Escherichia coli;
genetics;
metabolism;
Fungal Proteins;
biosynthesis;
genetics;
metabolism;
Genes, Synthetic;
Genetic Vectors;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Single-Strand Specific DNA and RNA Endonucleases;
biosynthesis;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2012;28(11):1388-1397
- CountryChina
- Language:Chinese
-
Abstract:
To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 degrees C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized the functional expression of NP1 in the prokaryotic system for the first time, and provided an alternative pathway for NP1 preparation.
