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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.



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Article Details

Issue: Vol 18 No 1 (2015)
Page No.: 52-62
Published: Mar 31, 2015
Section: Natural Sciences - Research article
DOI: https://doi.org/10.32508/stdj.v18i1.1034

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Creative Commons License

Copyright: The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 How to Cite
Phan, T. (2015). Intracellular expression and investigation of the possibility for purifying recombinant protein in Bacillus subtilis using reporter GFP. Science and Technology Development Journal, 18(1), 52-62. https://doi.org/https://doi.org/10.32508/stdj.v18i1.1034

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