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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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[1] Green N M. Avidin and streptavidin. Methods in Enzymol, 1990, 184: 51-67. [2] Hatakeyama K, Tanaka T, Sawaguchi M, et al. Microfluidic device using chemiluminescence and a DNA-arrayed thin film transistor photosensor for single nucleotide polymorphism genotyping of PCR amplications from whole blood. Lab Chip, 2009, 9: 1052-1058. [3] Howarth M, Takao K, Hayashi Y, et al. Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc Nat Acad Sci US, 2005, 102(21): 7583-7588. [4] Larsson C, Rodahl M, Höök F. Characterization of DNA immobilization and subsequent hybridization on a 2D arrangement of streptavidin on a biotin-modified lipid bilayer supported on SiO2. Anal Chem, 2003, 75: 5080-5087. [5] Matsunaga T, Maeda Y, Yoshino T, et al. Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micropolystyrene bead composites,‘Beads on Beads’. Anal Chem Acta, 2007, 597: 331-339. [6] 路静, 施前. 生物素标记法分析MCF-10A细胞质膜蛋白质. 复旦学报(医学版), 2010, 37(6): 710-714. Lu J, Shi Q. Analysis on plasma membrane proteins of MCF10A by cell surface biotinylation. Fudan Univ J Med Sci, 2010, 37(6): 710-714. [7] Bayer E A, Wilchek M. Protein biotinylation. Methods in Enzymol, 1990, 184: 138-160. [8] Fall R R. Analysis of microbial biotin protein. Methods in Enzymol, 1979, 62: 390-398. [9] Barker D F, Campbell A M. Genetic and biochemical characterization of the birA gene and its product:Evidence for a direct role of biotin holoenzyme sythetase in repression of the biotin operon in Escherichia coli. J Mol Biol, 1981, 146(4): 469-492. [10] Li Y F, Sousa R. Expression and purification of E.coli BirA biotin ligase for in vitro biotinylation. Prot Expr Purif, 2012, 82: 162-167. [11] Dorothy B, Kovaleva E, Schatz P J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci, 1999, 8: 921-929. [12] Gutternigg M, Rendic D, Voglauer R, et al. Mammalian cells contain a second nucleocytoplasmic hexosaminidase. Biochem J, 2009, 419(1): 83-90. [13] 刘琳, 徐, 蔡春梅, 等. 人己糖胺酶D的原核表达, 纯化及酶学特性研究. 中国生物工程杂志, 2012, 32(9): 28-33. Liu L, Xu Y, Cai CH M, et al. Expression, purification and enzymatic characteristics of human Hex D in E.coli. China Biotechnology, 2012, 32(9): 28-33. [14] Romier C, Jelloul M B, Albeck S, et al. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallographica Section D: Biological Crystallography, 2006, 62(10): 1232-1242. [15] Choi R E, Schulman H, Cronan J E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci, 2004, 13(11): 3043-3050. |
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