Expression and purification of autotoxin in sf9 insect cells
10.3969/j.issn.1005-1678.2017.01.008
- VernacularTitle:Autotaxin在sf9昆虫细胞的表达及纯化研究
- Author:
Fengmei ZHAO
;
Weiwei PAN
;
Pengcheng CAO
;
Yarui ZHAO
;
Yongji HE
;
Yi ZHAO
- Keywords:
Autotaxin;
sf9 insect cell;
Bac-to-Bac expression system;
lysophospholipase D activity
- From:
Chinese Journal of Biochemical Pharmaceutics
2017;37(1):34-38
- CountryChina
- Language:Chinese
-
Abstract:
Objective Production of autotoxin protein in sf9 insect cells with biological activity. Methods Autotaxin cDNA was cloned into pFastBacTMHTA from melanoma cell by extraction of total RNA using TRIzol method and RT-PCR. Bacmid-ATX is isolated from transformed competent bacterial DH10 which carries Bac genomic sequences and transfected into sf9 using lipofectamine 2000. Recombinant ATX virus was amplified in sf9 and further used for infection and expression of ATX protein. Two step purification product using HistrapTMHP and Hiload 16/600 Suerdex 200pg was determined for lysophospholipase D (lysoPLD) activity. Results Correct insertion of PCR fragment is confirmed by BamH I/Xho I digestion and sequencing. ATX virus can infect sf9 and induced enzymatic activity. Column purification and SDS-PAGE resulted 95% in purity and 6mg/liter in yield with significant lysoPLD activity. Conclusion ATX Baculovirus was successfully constructed that can infect sf9 cells and express active lysoPLD. Production of active ATX can be used for crystalography studies and screening for small pharmaceutical inhibitors.
