Recombinant BirA Biotin Ligase, CBD-tag (rC-BirA)
The extremely tight bond between biotin and avidin or streptavidin makes the biotin labeling of proteins a useful tool for many applications. BirA is the Escherichia coli biotin ligase that biotinylated at a specific site a lysine side chain within a 15 amino acid acceptor peptide (also known as the Avi tag).
As a complementary approach to in vivo biotinylation of Avi tag-bearing proteins, we developed a protocol to produce recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as thioredoxin and MBP fusions and was released from the corresponding fusion by TEV protease. The released ligase was separated from its carrier using a HisTrap HP column.
Rat Cholesterol ELISA ELISA | |||
BlueGene | |||
Goat Cholesterol ELISA ELISA | |||
BlueGene | |||
Mouse Cholesterol ELISA ELISA | |||
BlueGene |
We obtained 24.7 and 27.6 mg of BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be very active in catalyzing biotinylation in vitro. The described protocol provides an efficient means for the manufacture of BirA ligase that can be used for the biotinylation of different Avi tag-bearing substrates.
Rat Cholesterol ELISA ELISA | |||
E01A11128 | |||
Goat Cholesterol ELISA ELISA | |||
E01A46041 | |||
Mouse Cholesterol ELISA ELISA | |||
E01A19869 |
Extending (strepto) avidin-biotin technology for total protein purification, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein-free protein matrix.
purification and many other possible applications, the avidin-biotin (streptococcus) interaction is best when reversible. A mild enzymatic
The method of introducing a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins that carry a biotinylation tag has been developed. The optimal condition for eYcient biotin ligase (BirA) catalyzed in vitro distiobiotinylation of Escherichia coli in It has been established from 1 to 4 h by systematically varying substrate concentrations, reaction time, and pH.
Rat Cholesterol ELISA ELISA | |
E01A11128 | BlueGene |
Goat Cholesterol ELISA ELISA | |
E01A46041 | BlueGene |
Mouse Cholesterol ELISA ELISA | |
E01A19869 | BlueGene |
Human Cholesterol ELISA ELISA | |
E01A2368 | BlueGene |
Sheep Cholesterol ELISA ELISA | |
E01A98335 | BlueGene |
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 purify aYnity desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis at 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 analogs into recombinant proteins at a specific site.
conduct.