Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris.
- Author:
Fangxia CHEN
1
;
Peng CHEN
1
Author Information
- Publication Type:Journal Article
- Keywords: Northern Shrimp Mg²⁺/Ca²⁺-independent nuclease; Pichia pastoris; catalytic characteristics; secretory expression optimization
- MeSH: Animals; Codon; Electrophoresis, Polyacrylamide Gel; Endonucleases; biosynthesis; Glycoproteins; Hot Temperature; Penaeidae; enzymology; Pichia; metabolism; Recombinant Proteins; biosynthesis
- From: Chinese Journal of Biotechnology 2016;32(7):991-995
- CountryChina
- Language:Chinese
- Abstract: Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.
