N-terminal LysSN-His-tag improves the production of intracellular recombinant protein in Bacillus subtilis.

Choosing fusion tags to enhance the recombinant protein levels in the cytoplasm of Bacillus subtilis has been limited. Our previous study demonstrated that His-tag at the N-terminus could increase the expression levels of the low-expression gene egfp, while significantly reducing the high-expression genes gfp+ and bgaB in the cytoplasm of B. subtilis. In this study, we aimed to prove the potential of a fusion tag, the combination of the N-terminal domain of B. subtilis lysyl tRNA synthetase (LysSN) and His-tag with varying numbers of histidine (6xHis, 8xHis, 10xHis) by investigating their effects on the expression levels of egfp, gfp+ and bgaB in B. subtilis. For the low-expression gene, LysSN-xHis-tag could enhance the fluorescent intensity of EGFP 23.5 times higher than EGFP without a fusion tag, and 1.5 times higher than that fused with only His-tag. For high-expression genes, the expression level of BgaB and GFP+ was 2.9 and 12.5 times higher than that of His-tag, respectively. The number of histidines in LysSN-xHis-tag did not influence the expression levels of the high-expression genes but affected the expression levels of the low-expression gene.

Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis.

The influence of fusion tags to produce recombinant proteins in the cytoplasm of Bacillus subtilis is not well-studied as in E. coli. This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene (gfp+) and low expression gene (egfp) in the cytoplasm of B. subtilis cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the Pgrac212 in B. subtilis. The fusions of His-tags with GFP+ caused a reduction compared to the construct without His-tag. When three His-tags fused with egfp, the EGFP production levels were significantly increased up to 3.5-, 12-, and 15-fold. This study suggested that His-tag at the N-terminus could enhance the protein production for the low expression gene and reduce that of the high expression gene in B. subtilis.