Investigating the expression of GFP fused with HIS-TAG at N- or Cterminus using plasmid pHT253 and pHT254 in Bacillus subtilis

Abstract

pHT253 and pHT254 belong to the plasmid system carrying Pgrac100, a strong promoter, which were commercialized in the global market in 2012 have pushed forward the use of Bacillus subtilis as host for the production of recombinant proteins. Along with strong promoter Pgrac100, these two plasmids were added His-tag to before and after MCS (Multi Cloning Site) in pHT253 (Pgrac100-8xHis-MCS) and pHT254 (Pgrac100- MCS-8xHis), respectively, to facilitate purification steps. Depending on the farget protein, the user selects appropriate effective strategy of fusing His-tag, i.e. either into the N- or the C-temimal of protein. In this study, the GFP gene was used as an indicator gene to investigate the influence of the His-tag position on the protein expression in B. subtilis. Our results showed that the expression of GFP in B. subtilis significantly was reduced in the fusion form with His-tag at the N-terminal. Thiswouldbe important information for the selection of suitable vectors.