Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 991-995, 2016.
Article
in Chinese
| WPRIM
| ID: wpr-242280
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pichia
/
Recombinant Proteins
/
Codon
/
Glycoproteins
/
Penaeidae
/
Electrophoresis, Polyacrylamide Gel
/
Endonucleases
/
Hot Temperature
/
Metabolism
Type of study:
Diagnostic study
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2016
Type:
Article
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