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| One-Step Preparation of Biotinylated Protein Using Prokaryotic Expression System |
| XIE Xiao-li1, CHENG Jian-song1, WANG Xiao-min1, MEI Xiang-han1, LIU Lin1, LI Jing1,2 |
1. College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China;
2. Synergetic Innovation Center of Chemical Science and Engineering, Tianjin 300071, China |
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Abstract Traditionally, proteins were labeled with biotin in vitro via chemical modification which involved in chemical activation, dialysis and purification of biotin and target protein. This process was exhausting, and often required excessive target protein for labelling. In this study, using pCDFDuet-1 as a prokaryotic co-expression plasmid, the cDNA of hexD (Hexosaminidase D) containing 6x His and the DNA of BAP (biotin acceptor peptides) were fused by PCR and inserted into MCS1 (Multiple cloning site1). birA (Biotin ligase) gene cloned from E.coli Trans5α genome was inserted into MCS2. The constructed recombinant plasmid pCDFDuet-hexD-BAP-birA was then verified by sequencing and transformed into E. coli BL21 (DE3) pLysS. The cell was induced by adding 0.1 mmol/L IPTG and 80 μmol/L biotin, and HexD-BAP was expressed and purified via Ni-NTA affinity chromatography and ultrafiltration. The molecular weight (60 kDa) and the purity (> 90%) of the protein were verified via SDS-PAGE. Using anti-HexD and streptavidin-HRP as antibodies, western blots showed that the recombinant HexD-BAP was expressed and successfully labeled with biotin by co-expressed BirA. By using 4-MU-O-GalNAc (4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-galctopyranoside) as substrate, the activity of biotin labeled HexD-BAP was determined as 3.6 nmol/(min·μg), which has a good match of the activity of the unlabeled HexD (3.06 nmol/(min·μg)). These results demonstrated that via BirA and BAP co-expression, exogenous proteins could be expressed and labeled with biotin in vivo without altering their bioactivity. This system involves one-step transformation, expression and purification, which can be an efficient and useful tool for many biological studies such as immune-labeling and interactive protein trap.
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Received: 12 October 2013
Published: 25 January 2014
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