RT Journal Article
SR Electronic
T1 A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 367730
DO 10.1101/367730
A1 Jeff Granhøj
A1 Henrik Dimke
A1 Per Svenningsen
YR 2018
UL http://biorxiv.org/content/early/2018/07/11/367730.abstract
AB Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. The E. coli protein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. A randomly mutated BirA library was used to identify novel variants. Peptide sequences from 13 out of 14 tested proteins showed a strong enrichment for bacteria after 3-5 selection rounds. Moreover, a clone selected for biotinylation of a C-terminal of peptide from red-fluorescent protein TagRFP showed site-specific biotinylation. Thus, active BirA variants with novel specificity are effectively isolated with our bacterial display system and provides a basis for the development of BirA variants for site-selective biotinylation.