Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion

Protein Expression and Purification
Sau-Ching WuSui-Lam Wong

Abstract

A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method for recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. The availability of large amounts of quality BirA can be critical for in vitro biotinylation. We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation. BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample. We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E. coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature. Application of this novel BirA to protein purification via in vitro biotiny...Continue Reading

References

Apr 15, 1979·Analytical Biochemistry·K P HenriksonW L Maloy
Jan 1, 1990·Methods in Enzymology·R A Kohanski, M D Lane
Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
May 15, 1988·Analytical Biochemistry·M Wilchek, E A Bayer
Jun 11, 1985·Biochimica Et Biophysica Acta·A Piffeteau, M Gaudry
Aug 1, 1996·The Biochemical Journal·J Tucker, R Grisshammer
Aug 1, 1996·Biochemical and Molecular Medicine·K OhnoD Meruelo
Mar 6, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·J Turková
Apr 22, 1999·Protein Science : a Publication of the Protein Society·D BeckettP J Schatz
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·J S SmithS E Spence
Sep 2, 1999·Trends in Biochemical Sciences·A Chapman-Smith, J E Cronan
Oct 19, 2000·Methods in Enzymology·J A Bornhorst, J J Falke
Oct 19, 2000·Methods in Enzymology·M G Cull, P J Schatz
Feb 13, 2001·Analytical Biochemistry·K L EwaltM Krihak
Mar 17, 2001·Journal of Immunological Methods·D J Cahill

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Citations

Jan 22, 2013·Trends in Biochemical Sciences·Eva RieserHenning Walczak
May 29, 2012·Biochemical and Biophysical Research Communications·Mio ShibataShigetsugu Hatakeyama
Jun 21, 2007·Critical Reviews in Biotechnology·Parvinder KaurT Satyanarayana
Mar 1, 2006·International Journal of Biological Macromolecules·Aline TiratLukas Leder

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