Abstract
most popular model organisms for Gramnegative and Gram-positive bacteria. Though this Gram-positive bacterium has a great advantage – endotoxin-free, the use of B. subtilis for expression and purification is not much interested as compared to E. coli. The reason for this is the lack of information and technology platforms on the production of recombinant proteins in B. subtilis. In the previous studies, pHT vector system has been demonstrated to allow the high levels of recombinant protein expression in B. subtilis. In this study, we used GFP as a marker for intracellular expression in B. subtilis and examining the purification capability of the recombinant protein. The expression vector was designed with gfp gen fused to the gen encoding the N-terminus of LysS protein (lysSN), His-tag and specific cleavage site of TEV protease to enhance the expression of the target protein as well as contribute to the purification and removing fusion tag afterwards. The results showed that this vector allowed the effective expression of the fusion protein LysSN- 6xHis-TEV-GFP in B. subtilis, the target protein could be purified through Ni2+ column with a high purity and fusion tag could be completely removed by TEV protease. Recombinant GFP obtained after purification was determined the molecular weight by LCMS that exhibited the analogy with the natural GFP protein. This study showed a great potential of using pHT expression system with endotoxin-free B. subtilis as a host for intracellular expression and purification of recombinant proteins.
Highlights
Escherichia coli and Bacillus subtilis are the most popular model organisms for Gramnegative and Gram-positive bacteria. Though this Gram-positive bacterium has a great advantage – endotoxin-free, the use of B. subtilis for expression and purification is not much interested as compared to E. coli. The reason for this is the lack of information and technology platforms on the production of recombinant proteins in B. subtilis
PHT vector system has been demonstrated to allow the high levels of recombinant protein expression in B. subtilis
The expression vector was designed with gfp gen fused to the gen encoding the N-terminus of
Summary
Bacillus subtilis và Escherichia coli là hai mô hình nghiên cứu đối với vi khuẩn Gram dương và Gram âm, đồng thời cũng là hai hệ thống vi khuẩn được sử dụng nhiều trong lĩnh vực công nghệ protein tái tổ hợp. Với E. coli, B. subtilis tuy có nhiều ưu điểm hơn, nhất là không lẫn nội độc tố trong sản phẩm, nhưng các nghiên cứu cũng như công nghệ nền phục vụ việc biểu hiện và tinh chế protein tái tổ hợp ở vi khuẩn Gram dương này vẫn chưa được quan tâm rộng rãi. Một số nghiên cứu trước đây đã phát triển thành công hệ thống vector pHT cho phép biểu hiện protein tái tổ hợp trong B. subtilis một cách hiệu quả nhưng vẫn chưa tiến hành tinh chế và thu nhận protein mục tiêu. Ở nghiên cứu này, chúng tôi tiếp tục sử dụng hệ thống pHT trong B. subtilis để đánh giá khả năng biểu hiện nội bào của protein chỉ thị GFP sau khi đã dung hợp với một đuôi tinh chế, đồng thời thử nghiệm khả năng tinh chế protein mục tiêu. Từ khóa: Bacillus subtilis, LysSN, GFP, hệ thống biểu hiện pHT, sắc kí ái lực
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