Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

doi:10.1016/j.ab.2004.03.056

Analytical Biochemistry

Volume 331, Issue 2, 15 August 2004, Pages 340-348

https://doi.org/10.1016/j.ab.2004.03.056 Get rights and content

Abstract

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 1–4 h 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.

Section snippets

Construction of Bacillus subtilis expression vector for overproducing SAK-T-PFB

Plasmid pSAK-T-PFB is a derivative of pSAKPFB [24] which is a pWB980-based vector [26] for secretory production of staphylokinase (SAK) containing a C-terminal biotinylation tag (PFB). A nucleotide sequence encoding a 6 amino-acid peptide (LVPRGS) with a thrombin cleavage site was inserted between the nucleotide sequences encoding the C-terminal end of the linker and the N-terminal end of PFB (Fig. 1) in pSAKPFB to generate pSAK-T-PFB. Production of SAK-T-PFB is under the control of the B.

Establishment of the optimal conditions for desthiobiotinylation

The structure of desthiobiotin differs in a number of respects from biotin. While desthiobiotin retains the uredio ring of biotin, it misses the 5-member sulfur-containing thiophene ring (Fig. 2). As well, unlike biotin which has the pentanoic acid attached to the thiophene ring, a hexanoic acid is attached directly to the uredio ring in desthiobiotin. It is unsure how these structural differences would affect the efficiency for desthiobiotin to serve as a substrate for BirA. While the less

Discussion

Escherichia coli BirA ligase has been applied successfully to biotinylate engineered proteins carrying biotinylation tags [28], [32], [33], [34]. In this study, we demonstrate the feasibility to extend the application of E. coli BirA for site-specific desthiobiotinylation of recombinant proteins. In comparison with the biotin concentration normally used for biotinylation, the concentration of desthiobiotin used for desthiobiotinylation is at least 40 times more. Analysis of the structure of the