Expression, purification and characterization of focal adhesion kinase using a baculovirus system

Focal adhesion kinase (FAK) has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus was generated which contains the mouse FAK cDNA cloned into a histidine tag transfer vector. Synthesis of the immunoreactive recombinant protein (baculovirus focal adhesion kinase (BFAK) Mr approximately 125,000) in infected Sf9 cells was detected 23 h postinfection, with maximal accumulation occurring at 48 h postinfection. BFAK constituted 5.4% of total soluble protein in the insect cell lysate and represented 19 mg/liter culture (approximately 2 x 10(9) cells). The enzyme was active as a protein tyrosine kinase in both SF9 cells and in vitro. Purification to near homogeneity was achieved by nickel chelation chromatography. A yield of 5 mg of purified active BFAK was consistently produced from 1 liter of infected insect cells. BFAK tyrosine kinase activity was characterized physically using poly(Glu-Tyr) as a substrate. BFAK activity required the presence of a divalent cation and exhibited a preference for Mn2+ over Mg2+. Maximal tyrosine kinase activity was attained at pH 7.2. Steady-state kinetic analysis with respect to ATP concentration did not conform to simple Michaelis-Menten kinetics and exhibited a Hill coefficient of much less than 1. Km values for ATP using native and autophosphorylated BFAK were 6.7 +/- 1.0 and 4.3 +/- 0.2 microM, respectively. Kcat values were 13.9 +/- 1.9 and 8.9 +/- 0.3 nmol/min/mg BFAK. Steady-state kinetics with respect to the peptide substrate did fit the Michaelis-Menten equation and exhibited a Km value of 2.4 +/- 0.3 micro/ml.