In many cases, the analysis of a specific protein is hindered by the inability to purify large quantities of it from a native source. It's possible that proteins of interest are only found in trace amounts, or that attempts to purify them have been hampered by technical difficulties. DNA recombinant techniques have allowed researchers to overcome some of these limitations by producing large quantities of purified proteins from nonnative systems. Biotin labelling proteins with biotin is a useful tool for a wide range of applications because of the strong affinity between biotin and avidin or streptavidin. The biotin ligase from Escherichia coli, BirA, biotinylates a lysine side chain in a 15-amino acid acceptor peptide (also known as Avi-tag). We developed a method for producing recombinant BirA ligase for in vitro biotinylation of Avi-tag-bearing proteins. The target protein was expressed in both thioredoxin and MBP fusions, and the corresponding fusion was released by TEV protease. The HisTrap HP column was used to separate the free ligase from its carrier. In the case of thioredoxin and MBP fusion constructs, we obtained 24.7 and 27.6 mg BirA ligase per litre of culture, respectively. The recombinant enzyme was found to be extremely effective in vitro when it came to biotinylation. The procedure outlined here is an efficient way to make BirA ligase, which can be used to biotinylate a variety of Avi-tag-bearing substrates.