The BirA  biotin-protein ligase  adds d-biotin  covalently to  biotin-acceptor peptides/proteins  via an ATP

intermediate (biotinyl 5’-adenylate) in a highly efficient ａnd targeted  manner. The protein is encoded by

the birA gene  ａnd is purified using traditional  methods. The molecular weight of  the BirA biotin-protein

ligase is 33,500 Daltons.

Other  names  for   this  enzyme  include:   biotin  ligase;  biotin  operon   repressor  protein;  birA;   biotin

holoenzyme  synthetase;  biotin-[acetyl-CoA   carboxylase]  synthetase;

biotin-[acetyl-CoA-carboxylase]

ligase;  biotin-[acetyl-CoA carboxylase]  synthetase;  acetyl CoA  holocarboxylase  synthetase; acetyl  CoA

holocarboxylase synthetase; biotin:apocarboxylase ligase; biotin holoenzyme synthetase; HCS.

Source: E. coli

Enzyme Storage buffer: 50mM imidazole (pH 6.8), 50mM NaCl, 10% glycerol, 5mM dithiothreitol (DTT).

Storage conditions: The BirA-500 biotin-protein ligase kit  arrives on dry ice (or, in rare cases, ice brix)  via

overnight delivery and  should be immediately  stored at -80°C. After  an enzyme vial is  thawed it should

be stored  at 4°C if  it is to  be re-used  in the near  future. For long-term  storage keep  at -80°C. A  vial of

thawed enzyme can be re-frozen by dropping into liquid nitrogen before storing again at -80°C.

The other  kit components, BiomixA,  BiomixB and  BIO-200, can be  stored long term  at -20°C.  For short

term storage 4°C  is fine for  the BiomixA ａnd BIO-200.  The BiomixB should  always be stored at  -20°C or

colder ａnd thawed immediately before use.  There is no problem in thawing ａnd re-freezing any of  these

mixtures repeatedly.

Stability: BirA  biotin-protein ligase retains  >80% of its  activity when stored  for one month  at 4°C.  Long

term  storage should  be  at -80°C.  Refreezing  the  enzyme is  possible  but will  reduce  its activity  upon

thawing  again  by 10%  each  time.  Therefore  we  don’t recommend  repeated  thawing  and  refreezing

cycles with the BirA.

Concentration: 1.0 mg/mL by A280 (Extinction coefficient; 1.349AU at 280nm λ=1mg/mL BirA).

Purity: >99% by Coomassie staining.

Activity: a minimum of 5,000 Units/µg of BirA

Definition  of  Activity:  One  (1)  Unit  is  the  amount  of  enzyme  that  will  biotinylate  1pmol  of  biotin-

acceptor  peptide  substrate*  in  30  minutes  at  30°C  using  the  reaction  buffers  provided  and  40μM

peptide substrate final concentration.

* The peptide  substrate used in the enzyme  assays is a 15-mer  variant of sequence #85  identified by P.

Schatz (1).