Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Avraham S, Jiang S, Ota S, Fu Y, Deng B, Dowler LL, White RA, Avraham H (1995) Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product. J. Biol. Chem. 270: 1833–1842.
Ayerapetov MK, Lee S, Sun G (2003) Expression, purification and biochemical characterization of Chk, a soluble protein tyrosine kinase. Protein Expr. Purif. 29: 148–155.
Bougeret C, Jiang S, Keydar I, Avraham H (2001) Functional analysis of Csk and CHK kinases in breast cancer cells. J. Biol. Chem. 276: 33711–33720.
Bretthauer RK, Castellino FJ (1999) Glycosylation of Pichia pastoris derived proteins. Biotechnol. Appl. Biochem. 30: 193–200.
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia patoris. FEMS Microbiol. Rev. 24: 45–66.
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Molec. Biotechnol. 16: 23–52.
Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54: 741–750.
Higgins DR, Cregg JM (1998) Introduction to Pichia pastoris. Meth. Mol. Biol. 103: 1–15.
McShan GD, Zagozdzon R, Park SY, Zrihan-Licht S, Fu Y, Avraham S, Avraham H (2002) Csk Homologous Kinase associates with RAFTK/pyk2 in breast cancer cells and negatively regulates its activation and breast cancer cell migration. Int. J. Oncol. 21: 197–205.
Romanos M (1995) Advances in the use of Pichia pastoris for highlevel expression. Curr. Opin. Biotechnol. 6, 527–533.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in themethylotrophic yeast Pichia pastoris. Gene 190: 55–62.
Yamaguchi N, Nakayama Y, Urakami T, Suzuki S, Nakamura T, Suda T, Oka N (2001) Over-expression of Csk homologous kinase (Chk tyrosine kinase) induces multinucleation: a possible role for chromosome-associated Chk in chromosome dynamics. J. Cell. Sci. 114: 1631–1641.
Zrihan-Licht S, Deng B, Yarden Y, McShan g, Keydar I, Avraham H (1998) Csk homologous kinase, a novel signaling molecule, directly associates with the activated ErbB-2 receptor in breast cancer cells and inhibits their proliferation. J. Biol. Chem. 273: 4065–4072.
Zrihan-Licht S, Lim J, Keydar I, Sliwkowski MX, Groopman JE, Avraham H (1997) Association of csk-homologous kinase (CHK) (formerly MATK) with HER-2/ErbB-2 in breast cancer cells. J. Biol. Chem. 272: 1856–1863.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Murthy, T. Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification. Biotechnology Letters 26, 443–449 (2004). https://doi.org/10.1023/B:BILE.0000018265.25289.6d
Issue Date:
DOI: https://doi.org/10.1023/B:BILE.0000018265.25289.6d