Purification and characterization of human Syk produced using a baculovirus expression system.
The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling via a variety of immune and nonimmune cell receptors. Syk is activated in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain-containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells. Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10 microM and the peptide substrate was 3 microM. The recombinant protein also showed similar biochemical characteristics to the native protein isolated from B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by micro-calpain. We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.
Baldock
D
D
Respiratory Disease Therapeutic Area, Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex, RH12 5AB, United Kingdom. Darren.Baldock@pharma.novartis.com
Graham
B
B
Akhlaq
M
M
Graff
P
P
Jones
C E
CE
Menear
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D000072377:Syk Kinase
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase