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Fujinami sarcoma virus P140 proteolysis and peptide purification

University of British Columbia

Fujinami sarcoma virus encodes a 140/000 m.w. polypeptide (P140) which has been correlated as the agent of transformation in host chicken fibroblasts and mammalian fibroblasts. To conclusively identify the role of P140 in the transformation process it will be necessary to obtain intact/ purified P140. The availability of an antibody monoclonally specific to the N-terminal gag encoded portion of P140 suggested a one-step immunoaffinity purification of P140. After purification of the antibody out of mouse ascites fluid, by 50% ammonium sulfate fractionation and ion exchange chromatography, antibody was linked to a Sepharose 4-B matrix activated with cyanogen bromide. The anti-pl9 affinity matrix bound intact P140 as a doublet relative to a polyclonal anti-pl9. Chaotropic agents, high pH and low pH treatments all failed to elute the bound P140 from the affinity matrix. Failing the purification of intact P140 a method of partial proteolysis was used to produce varying sized fragments of P140/ with the goal of purifying these fragments for further work on the role of P140. Trypsin alone in a limited proteolysis produced small, unstable peptides too close in size distribution to be effectively purified. Chymotrypsin alone produced a broad range of more stable peptides, with a predominance of a 45,000 m.w. peptide. Chymotrypsin-trypsin consecutive proteolysis produced a very stable 35,000 m.w. peptide. Gel filtration of the chymotryptic peptides was ineffective as the peptides coraplexed and were not fractionated. Ion exchange chromatography fractionated the complexing chymotryptic peptides, making possible the purification of these peptides. The stable 45,000 m.w. peptide retained some kinase activity, as it phosphorylated the substrate enolase, similar to but less intense than intact P140. A 30,000 m.w. peptide only phosphorylating after ion exchange did not phosphorylate enolase.

Text

2010-05-08

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http://hdl.handle.net/2429/24487

Microbiology and Immunology

University of British Columbia

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