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.
Withers
B E
BE
Department of Cancer Research, Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48105, USA.
Keller
P R
PR
Fry
D W
DW
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D015815:Cell Adhesion Molecules; D002614:Chelating Agents; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; C027606:L-glutamic acid-L-tyrosine copolymer; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D008274:Magnesium