Biotin (Vitamin B7) is an essential coenzyme that is required by all organisms. Biotinylation is attaching biotin to a protein carried out by biotin protein ligases (BPLs). Biotin binds Streptavidin and Avidin with very high affinity and this affinity is exploited in various assay formats including biophysical approaches like surface plasmon resonance (SPR).<\/p>\n
Proteins can be specifically biotinylated using the natural specificity of the E. coli protein BirA. BirA specifically biotinylates a single lysine (K122) on the BCC subunit of acetyl-CoA carboxylase. Thus, adding this acceptor peptide sequence to a protein allows specific biotinylation using recombinant BirA. Biotinylation of recombinant proteins can be carried out by co-expressing the target protein along with BirA. To achieve this aim, we designed a series of constructs for intracellular co-expression in E. coli, and intracellular and secreted co-expression in HEK\/CHO\/Expi293F and insect cells. Biotin is added to a final concentration of 4\u00b5M at the point of IPTG induction (for E. coli) or the point of co-transfection\/co-infection (for HEK\/CHO & insect respectively).<\/p>\n
Biotinylation was confirmed across all expression systems using a combination of intact mass spectrometry, peptide mapping and gel shift assay as part of the routine quality control package. The data for the E3 ligases Cereblon\/DDB1 and VCB complex (VHL, elongin B and elongin C), and an intrinsically disordered transcription factor show that biotinylation of recombinant proteins can be carried out by co-expressing the target protein along with BirA. This can be done across all the expression systems we currently offer at Peak Proteins (E. coli, mammalian (HEK\/CHO\/Expi293F), insect) and we regularly use this technology to biotinylate proteins for our clients for a range of uses including compound screening and SPR. This offers significant advantages such as savings in time and material costs.<\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"featured_media":2831,"template":"","category":[680,703,702],"class_list":["post-4110","poster","type-poster","status-publish","has-post-thumbnail","hentry","category-protein-and-structure","category-protein-characterisation","category-protein-expression-and-purification"],"acf":[],"yoast_head":"\n