Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.
The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.
Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. liy10@uthscsa.edu
Yifeng
Y
Li
Rui
R
Sousa
D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013879:Thioredoxins; D001710:Biotin; D010450:Endopeptidases; C099488:TEV protease; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli