Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro.
To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.
Wu
Sau-Ching
SC
Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4.
Wong
Sui-Lam
SL
D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D001710:Biotin; C004749:desthiobiotin