Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification.
Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors. The kinase domain of CHK (amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated. Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation. The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active. A simple plate assay to identify colonies of P. pastoris expressing the recombinant protein is also presented.
Murthy
T V S
TV
Division of Experimental Medicine, Harvard Institutes of Medicine, 4-Blackfan Circle, Boston, MA 02115, USA. tmurthy@hms.harvard.edu
D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src)