Year : 2015 | Volume
: 4 | Issue : 1 | Page : 149-
Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications
Mohammad Hossein Etemadzadeh1, Arash Arashkia1, Farzin Roohvand1, Dariush Norouzian2, Kayhan Azadmanesh1
1 Department of Virology, Pasteur Institute of Iran, Tehran, Iran
2 Department of Pilot Biotechnology, Pasteur Institute of Iran, Tehran, Iran
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.
Dr. Kayhan Azadmanesh
Department of Virology, Pasteur Institute of Iran, 69 Pasteur Avenue, Kargar Avenue, Tehran - 1316943551
|How to cite this article:|
Etemadzadeh MH, Arashkia A, Roohvand F, Norouzian D, Azadmanesh K. Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications.Adv Biomed Res 2015;4:149-149
|How to cite this URL:|
Etemadzadeh MH, Arashkia A, Roohvand F, Norouzian D, Azadmanesh K. Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications. Adv Biomed Res [serial online] 2015 [cited 2020 Jul 10 ];4:149-149
Available from: http://www.advbiores.net/article.asp?issn=2277-9175;year=2015;volume=4;issue=1;spage=149;epage=149;aulast=Etemadzadeh;type=0