Yeast expression and application of an antifreeze protein from the desert beetle Microdera punctipennis.
- Author:
Shanshan MENG
;
Wenping CAI
;
Ji MA
- Publication Type:Journal Article
- MeSH:
Animals;
Antifreeze Proteins;
biosynthesis;
Blotting, Western;
Cold Temperature;
Coleoptera;
Cryoprotective Agents;
chemistry;
Electrophoresis, Polyacrylamide Gel;
Freezing;
Insect Proteins;
biosynthesis;
Mice;
Pichia;
metabolism;
Sf9 Cells
- From:
Chinese Journal of Biotechnology
2015;31(8):1255-1265
- CountryChina
- Language:Chinese
-
Abstract:
Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.
