Biotin peac5 maleimide

Yeast F₁ was genetically engineered using the QuikChange method (Agilent Tech.) to include two surface cysteine residues, γD101C and γE189C and a His₆-tag on the amino end of the β-subunit, and was purified as described46 (link). The cysteine residues were biotinylated by incubating: 10 mM (3.5 mg/ml) F₁, 20 mM Biotin-PEAC5-maleimide (6-{N′-[2-(N-Maleimido)ethyl]-N-piperazinylamido}hexyl D-biotinamide, hydrochloride, Dojindo Laboratories), 50 mM HEPES, pH 8.0, 2 mM ATP, 2 mM MgSO₄; for 30 min at 23°C, quenching with 100 mM DTT, and desalting on a centrifuge column containing Biogel P6 resin (Biorad). The protein concentration was adjusted to 0.4 mg/ml in buffer containing 50% glycerol, flash frozen and stored in liquid N₂. This modification does not alter the activity of the enzyme.

For biotinylation of Cys-apoA-I, 10-fold molar excess of tris(2-carboxyethyl)phosphine HCl (TCEP, Thermo Fisher Scientific) was added to a solution of Cys-apoA-I in PBS, and the mixture was incubated for 1.5 h at room temperature to generate sulfhydryl groups. Then, 10-fold molar excess of biotin-PEAC₅-maleimide (Dojindo, Kumamoto, Japan), dissolved in dimethyl sulfoxide (DMSO), was added to the reduced Cys-apoA-I in PBS (1 ml), and the mixture was incubated overnight at 4 °C. For biotinylation of plasma apoA-I, recombinant wild-type and 1–83 fragment of apoA-I, and mAbs, three- (for mAbs) or 10-fold (for apoA-I proteins) molar excess of EZ-Link NHS-LC-Biotin (Thermo Fisher Scientific), dissolved in DMSO, was added to the target protein in PBS (500 μl), and the mixture was incubated for 2 h at 4 °C. After these reactions, unreacted reagents were removed by extensive dialysis at 4 °C in PBS. The degree of labeling was determined by SensoLyte HABA biotin quantitation kit (AnaSpec, Fremont, CA).