Method of producing heterodimer derivative of protein...

Details Method of producing heterodimer derivative of protein... Method of producing heterodimer derivative of protein...

2005-11-16

2010-06-29

Nashed, Nashaat T (Department: 1656)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

Reexamination Certificate

active

07745191

ABSTRACT:
The purpose of the invention is to provide an activated protein phosphatase 2A (PP2A) in large quantities with high purity by a genetic engineering and to provide a method for producing a heterodimer derivative of PP2A which comprises infecting insect cultured cells with a baculovirus in which a cDNA encoding the catalytic subunit of PP2A carrying a first tag is integrated together with another baculovirus in which a cDNA encoding the A subunit of PP2A carrying a second tag is integrated, incubating the infected cells, disrupting the incubated cells to obtain a disrupted cell suspension, and then purifying the disrupted cell suspension with a solid phase carrying a substance capable of binding to the first tag and another solid phase carrying a substance capable of binding to the second tag, characterized in that the insect cells infected with the baculovirus are incubated at a temperature of from 18 to 22° C.

REFERENCES:
patent: 2003 250598 (2003-09-01), None
patent: WO 2006/048923 (2006-05-01), None
Ikehara et al. Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta. Protein Expression and Purification 45 (2206) 150-156 (available on line Jul. 1, 2005).
Myles T. et al., “Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells”, Biochem. J., vol. 657, p. 225 (2001).
S. Reuveny, et al., “Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures”, Applied Microbiology and Biotechnology, Springer Verlag, vol. 38, No. 5, XP000876997, Feb. 1, 1993, pp. 619-623.
Michael Donaldson, et al., “Glycosylation of a Recombinant Protein in the Tn5B1-4 Insect Cell Line: Influence of Ammonia, Time of Harvest, Temperature, and Dissolved Oxygen”, Biotechnology and Bioengineering, vol. 63, No. 3, XP009118107, May 5, 1999, pp. 255-262.

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