Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation

Abstract

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.

Fig. 1

Schematic representation of the two constructs in which the target protein is expressed as a fusion protein with (A) thioredoxin or (B) MBP.

Fig. 2

Expression/cloning region of (A) original pET-28a and (B) its modified version, in which a sequence encoding MBP was inserted between the NdeI and the BamHI sites. This modification allows the vector to be used for MBP fusion expression.

Fig. 3

Expression and purification of BirA fusion proteins as followed by SDS-PAGE (12%). (A) The target protein was expressed as a thioredoxin fusion. Lane 1, cell lysate supernatant; Lane 2, cell lysate pellet (resuspended in 8 M urea); Lane 3, imidazole eluate (Ni-NTA resin purified protein); Lane 4, size-exclusion chromatography purified protein; Lane 5, protein standards. (B) The target protein was expressed as an MBP fusion. Lane 1, cell lysate supernatant; Lane 2, cell lysate pellet (resuspended in 8 M urea); Lane 3, affinity and size-exclusion chromatography purified fusion protein; Lane 4, protein standards.

Fig. 4

Expression and purification of TEV protease as followed by SDS-PAGE (12%). Lane 1, cell lysate supernatant; Lane 2, cell lysate pellet (resuspended in 8 M urea); Lane 3, cell lysate supernatant after binding with Ni-NTA resin (unbound proteins); Lane 4, imidazole eluate (Ni-NTA resin purified protein); Lane 5, protein standards.

Fig. 5

(A) Cleavage efficiency of the thioredoxin-BirA fusion protein with different amounts of TEV protease as followed by SDS–PAGE (12%). The reactions were allowed to proceed for 16 h at room temperature. Lane 1–6, 0, 2, 4, 6, 8, 10 μg of TEV protease was added to 1 mg of fusion protein, respectively; Lane 7, protein standards. (B) Separation of the target protein from the carrier thioredoxin by using a HisTrap HP column (5 ml). Lane 1–3, fractions that flowed through the column and which contain the target protein; Lane 4–5, factions that bound to the column and eluted with increasing concentration of imidazole, Lane 6, protein standards.

Fig. 6

(A) thioredoxin and (B) MBP fusion cleavage and target protein purification as followed by SDS-PAGE (12%). Lane 1, corresponding fusion proteins purified by affinity and size-exclusion chromatography; Lane 2, reaction mixture after TEV protease cleavage; Lane 3, purified BirA; Lane 4, protein standards. The position of bands corresponding to the released BirA is indicated by an asterisk.

Fig. 7

(A) Schematic representation and (B) size-exclusion chromatography elution profile of the Avi-tagged thioredoxin. Inset shows SDS-PAGE analysis of the major peak component.

Fig. 8

(A) Biotinylation at different enzyme to substrate molar ratios. Reactions were allowed to proceed for 14 h at room temperature. Lanes 1–7: 0, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μM of recombinant BirA was added to 30 μM of substrate, respectively; Lane 8, protein standards. (B) Biotinylation reaction monitored as a function of time (enzyme to substrate molar ratio was 1:100). Lane 1–7, aliquots taken from the reaction at 0, 2, 4, 6, 8, 10 and 12 h, respectively; Lane 8, protein standards.

Fig. 9

Capture of biotin-labeled thioredoxin by Streptavidin Sepharose to confirm biotinylation. Lane 1, unlabeled thioredoxin; Lane 2, labeled thioredoxin; Lane 3, solution of unlabeled thioredoxin after incubation with Streptavidin Sepharose; Lane 4, solution of biotin-labeled thioredoxin after incubation with Streptavidin Sepharose (the faint band on the gel is BirA as indicated); Lane 5, biotin eluate that recovers the biotin-labeled thioredoxin captured by the resin; Lane 6, protein standards.

Fig. 10

Activity assay for thioredoxin-BirA and MBP-BirA fusion proteins. Lane 1, negative control of the biotinylation reaction, which contains all the reagents except for the biotin ligase; Lane 2–4, biotinylation by released BirA (positive control), thioredoxin-BirA and MBP-BirA, respectively (enzyme to substrate molar ratio was 1:50 in all three cases and the reactions were proceeded at room temperature for 4 h); Lane 5, protein standards.