Abstract
Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.
Similar content being viewed by others
Abbreviations
- h.p.i.:
-
hours post infection
- pNPP:
-
p-nitro-phenyl phosphate
- PTP:
-
protein tyrosine phosphatase
- RK682:
-
3-hexadecanoyl-5-hydroxymethyl-tetronic acid
- RU:
-
relative units
References
Dadke S., Kusari J., Chernoff J. 2000. Down-regulation of insulin signaling by protein-tyrosine phos-phatase 1B is mediated by an N-terminal binding region. J. Biol. Chem. 275, 23642–23647.
Sorenson C.M., Sheibani N. 2002. Altered regulation of SHP-2 and PTP 1B tyrosine phosphatases in cystic kidneys from bcl-2 -/-mice. Am. J. Physiol. Renal Phys-iol. 282, F442–F450.
Liu F., Sells M.A., Chernoff J. 1998. Protein tyrosine phosphatase 1B negatively regulates integrin signaling. Curr.Biol. 8, 173–176.
Dube N., Bourdeau A., Heinonen K.M., Cheng A., Loy A.L., Tremblay M.L. 2005. Genetic ablation of protein tyrosine phosphatase 1B accelerates lympho-magenesis of p53-null mice through the regulation of B-cell development. Cancer Res. 65, 10088–10095.
Frangioni J.V., Beahm P.H., Shifrin V., Jost C.A., Neel B.G. 1992. The non-transmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell. 68, 545–560.
Yang J., Liang X., Niu T., Meng W., Zhao Z., Zhou G.W. 1998. Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1. J. 1. J. Biol. Chem. 273, 28199–28207.
Yang J., Liu L., He D., Song X., Liang X., Zhao Z.J., Zhou G.W. 2003. Crystal structure of human protein-tyrosine phosphatase SHP-1. J. Biol. Chem. 278, 6516–6520.
Moeslein F.M., Myers M.P., Landreth G.E. 1999. The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B. J. Biol. Chem. 274, 26697–26704.
Huang P., Ramphal J., Wei J., Liang C., Jallal B., McMahon G., Tang C. 2003. Structure-based design and discovery of novel inhibitors of protein tyrosine phosphatases. Bioorg. Med. Chem. 11, 1835–1849.
Fujii G.H., Morimoto A.M., Berson A.E., Bolen J.B. 1999. Transcriptional analysis of the PTEN/MMAC1 pseudogene, psiPTEN. Oncogene. 18, 1765–1769.
Sorio C., Mendrola J., Lou Z., LaForgia S., Croce C.M., Huebner K. 1995. Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleo-sides. Cancer Res. 55, 4855–4864.
Ikehara T, Shinjo F., Ikehara S., Imamura S., Yasu-moto T. 2006. Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta. Protein Expr. Purif. 45, 150–156.
MylesT., Schmidt K.,EvansD.R.,CronP., Hemmings B.A. 2001. Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overex-pression in insect cells. Biochem. J. 357, 225–232.
Zhang Z.Y. 1998. Protein-tyrosine phosphatases: Biological function, structural characteristics, and mechanism of catalysis. Crit. Rev. Biochem. Mol. Biol. 33, 1–52.
Denu J.M., Dixon J.E. 1998. Protein tyrosine phosphatases: Mechanisms of catalysis and regulation. Curr. Opin. Chem. Biol. 2, 633–641.
Cool D.E., Tonks N.K., Charbonneau H., Fischer E.H., Krebs E.G. 1990. Expression of a human T-cell pro-tein-tyrosine-phosphatase in baby hamster kidney cells. Proc. Natl.Acad. Sci. USA. 87, 7280–7284.
Cromlish W.A., Payette P., Kennedy B.P. 1999. Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculov-iruses. Biochem. Pharmacol. 58, 1539–1546.
Author information
Authors and Affiliations
Corresponding author
Additional information
The article is published in the original.
Rights and permissions
About this article
Cite this article
Sundaram, S., Tiwari, P., Saini, S. et al. Baculovirus expression system: An alternative for producing catalytically active human PTP-1B. Mol Biol 44, 473–478 (2010). https://doi.org/10.1134/S0026893310030179
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026893310030179