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Year : 2015  |  Volume : 4  |  Issue : 1  |  Page : 149

Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications

1 Department of Virology, Pasteur Institute of Iran, Tehran, Iran
2 Department of Pilot Biotechnology, Pasteur Institute of Iran, Tehran, Iran

Correspondence Address:
Dr. Kayhan Azadmanesh
Department of Virology, Pasteur Institute of Iran, 69 Pasteur Avenue, Kargar Avenue, Tehran - 1316943551
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2277-9175.161576

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Background: The key enzyme in biotin-(strept) avidin systems, Escherichia coli BirA biotin ligase, is currently obtained by overexpression of the long protein-tagged versions of the gene to prevent its toxic effect in E. coli. Herein we describe a rather simple and efficient system for expression of E. coli BirA without the application of long-tag proteins. Materials and Methods: The coding sequence of BirA gene was isolated by polymerase chain reaction using DNA extract of E. coli-DH5α as template. BirA amplicon harboring a GS-linker at its C-terminal was cloned into NdeI-XhoI sites of pET24a(+) vector under control of T7 promoter and upstream of the vector-derived 6xHis-tag. pET24-BirA transformed BL21-cells were induced for protein expression by IPTG and analyzed by SDS-PAGE and Western blotting. Protein expression yields were assessed by image analysis of the SDS-PAGE scans using ImageJ software. Result: Agarose gel electrophoresis indicated proper size of the BirA gene amplicon (963 bp) and accuracy of the recombinant pET24-BirA construct. Sequence alignment analysis indicated identical sequence (100%) of our isolate with that of the standard E. coli-K12 BirA gene sequence (accession number: NC_000913.3). SDS-PAGE and Western blot results indicated specific expression of the 36.6 kDa protein corresponding to the BirA protein. Image analysis estimated a yield of 12% of total protein for the BirA expression. Conclusions: By application of pET24a(+) we achieved relatively high expression of BirA in E. coli without application of any long protein-tags. Introduction of the present expression system may provide more readily available source of BirA enzyme for (strept) avidin-biotin applications and studies.

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