title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications.
abstract: 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.
  
  
  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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Etemadzadeh
authors.affiliation: Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Arashkia
authors.affiliation: Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Farzin
authors.initials: F
authors.lastname: Roohvand
authors.affiliation: Department of Pilot Biotechnology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Dariush
authors.initials: D
authors.lastname: Norouzian
authors.affiliation: Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Kayhan
authors.initials: K
authors.lastname: Azadmanesh
keywords: Biotin–(strept) avidin; BirA; E. coli; biotin ligase; pET24a(+)