title: [Prokaryotic expression, purification and enzymatic properties of nuclease P1].
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.
authors: College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Yanan
authors: Y
authors: Wang
authors: Aiyun
authors: A
authors: Wei
authors: Meiyan
authors: M
authors: Wang
authors: Xiaobin
authors: X
authors: Wei
authors: Chao
authors: C
authors: Zhang
authors: Liwei
authors: L
authors: Shan
authors: Sanhong
authors: S
authors: Fan
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; C526100:Nuclease P1, Penicillium citrinum; D015719:Single-Strand Specific DNA and RNA Endonucleases