Expression of recombinant major surface protein 5 of Anaplasma marginale (A. marginale) at different temperatures

Abstract

Background : Bovine anaplasmosis is an important tick-borne disease caused by Anaplasma marginale                  (A. marginale) and infected in ruminants, mostly in cattle. This disease occurs in tropical and subtropical regions including Thailand and causes a major problem to livestock productions. The major surface protein 5 (MSP5) is one of outer membrane protein of A. marginale which as an immunodominant protein encoded by a single gene and also highly conserved gene.

Objective : The aim of this study was to optimize the conditions for the expression of recombinant major surface protein 5 (rMSP5) of A. marginale.

Methods : The msp5 gene of A. marginale was cloned into the pET100/D-TOPO^R vector to produce an pET100-msp5-6xHis fusion gene construct. The recombinant proteins were expressed by the plasmids in Escherichia coli
host strain BL21 star^TM (DE3) at different temperatures (16, 25 and 37 C) for 6 h. The proteins were analyzed by SDS-PAGE and confirmed the target protein by Western blotting using antisera against His.

Results : After induction with 0.1 mM of Isopropyl -D-1-thiogalactopyranoside (IPTG) at different temperatures for protein expression, the protein was not produced at 6 h for 16C. On the other hand, the rMSP5 protein was
produced at 25 and 37C for 2-6 h but the expressive protein at 25C showed lower yield than that at 37C.

Conclusion : In this study, the best condition for rMSP5 protein expression was cultured at 37C for 4 h. The protein was identified as the rMSP5 at the molecular weight of 26 kDa.