Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera

doi:10.3969/j.issn.1004-1524.2017.07.15

›› 2017, Vol. 29 ›› Issue (7): 1166-1171.DOI: 10.3969/j.issn.1004-1524.2017.07.15

• Plant Protection • Previous Articles Next Articles

Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera

DU Xinkai, REN Juan, HU Jun, WANG Changgao, LIN Jianguo, CAI Jun, DU Xin^*

Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China

Received:2017-02-22 Online:2017-07-20 Published:2017-07-24

Abstract

Abstract: Sterol carrier protein 2 (SCP-2) plays important physiological roles in sterols absorption and transport in insects. In this study, the recombinant expression plasmid pET-22b/HaSCP2 was constructed and transformed into E. coli BL21 (DE3) for inducible expression. Induction conditions of the IPTG concentrations, the induction time and temperature were optimized. The recombinant HaSCP-2 was purified using Ni²⁺-affinity column, and the protein functions of HaSCP-2 was tested by competitive displacement assay. SDS-PAGE analysis suggested that the molecular weight of the recombinant protein was about 16 ku, and the optimal expression conditions were at the temperature of 25 ℃ and at the final concentration of 0.2 mmol·L^-1 IPTG for 10 h. Fluorescent probe 8-aniline-1-naphthalenesulfonic acid (1,8-ANS) was used for competitive analysis, and 1,8-ANS was replaced by cholesterol bound to HaSCP-2 with a half effective concentration (EC₅₀) of 29.03 μmol·L^-1. This study provides good opportunity for further study on the function of HaSCP-2 and the screening of HaSCP-2 specific inhibitors.

Key words: Helicoverpa armigera, sterol carrier protein-2, prokaryotic expression, prokaryotic purification, prokaryotic optimization

CLC Number:

S435

DU Xinkai, REN Juan, HU Jun, WANG Changgao, LIN Jianguo, CAI Jun, DU Xin. Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera[J]. , 2017, 29(7): 1166-1171.

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Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera

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