Analysis of the tyrosine protein kinase p56lck expressed as a glutathione S-transferase fusion protein in Spodoptera frugiperda cells.
A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.
Spana
C
C
Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
O'Rourke
E C
EC
Bolen
J B
JB
Fargnoli
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D010751:Phosphopyruvate Hydratase