International Journal of Applied Microbiology and Biotechnology Research
ISSN: 2053-1818
Vol. 6(2), pp. 22-31, February, 2018

High-level expression of biotin ligase BirA from Escherichia coli K12 in Pichia pastoris KM71

Qingqing Li, Qiang Lu, Xiaojian Ma, Yaping Wang, Mengjie Dong, Zhen Zhang and Lixin Ma*

Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People’s Republic of China.

*To whom correspondence should be addressed. E-mail:

Received 12 January, 2018; Received in revised form 30 January, 2018; Accepted 02 February, 2018.


BirA, Biotinylation, Heterologous expression.

BirA is a biotin ligase from Escherichia coli that has been widely used in studying the interaction between avidin or streptavidin with its avi-tagged substrates, in which BirA adds a biotin molecular to a 15-amino acid acceptor peptide that is also known as avi-tag. The present study was aimed at obtaining high-level of BirA biotin ligase expression in Pichia pastoris KM71. In this study, the DNA coding sequence of BirA was amplified from E. coli K12. This novel gene was cloned into a lab-derived pHBM905A vector and transformed into P. pastoris KM71 for secretary expression. The expression of BirA was then identified by SDS–PAGE and Western blot analysis. The yield of the BirA reached approximately 103.5 mg/L after a 6-d induction in shake flasks, and 583.1 mg/L after 144 h of induction in 5 L fermenters. The expression levels obtained in this study were much higher than those currently reported, in which the heterologous expression of BirA with thioredoxin and MBP fusions in E. coli were 24.7 and 27.6 mg/L, respectively. The recombinant BirA exhibited high activity of catalyzing biotinylation in vitro with specific activity of 2291 U/μL. To the best of our knowledge, this is the first report of high-level BirA expression in P. pastoris KM71.

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