Interference No. 102,572
(CR-38).
(19) Coinventor Wetzel, a senior scientist at Genentech, testified that he had some
success in folding other recombinant proteins and his help was enlisted on the project.
Wetzel, ¶ 5 (CR 19-20). He and Perry, a research associate in his lab, began working on
the project in January, 1983. Wetzel, ¶ 6 (CR-20) , and Perry, ¶ 2
(CR-21-22 ). Initially they attempted to isolate and purify the heavy and light chains
produced in two different E. coli strains from cell pastes received by Mumford. Wetzel, ¶7
(CR-20).
17
(20) Perry testified that their strategy to refold the heavy and light chains from
singly transformed bacteria was to first purify the refractile bodies from the bacteria,
solubilize the protein in denaturant, followed by sulfitolysis. The chains would then be
further purified by S300 gel filtration chromatography and possibly DEAE ion-exchange
chromatography. The plan was to then reconstitute the antibodies by folding the heavy
chain first, adding it to the light chain, allowing both chains to fold together and then oxidize
the disulfide bonds. Perry, ¶ 3 (CR-22). She testified that they tried this strategy and
found a loss of heavy chain protein after the removal of the denaturant by dialysis into
native buffer. In order to alleviate proteolysis, they tried adding PMSF, EDTA, EGTA, and
altering pH and temperature. Protease was found to be inactivated by addition of PMSF,
17Other than referring to January, 1983, Perry, in her testimony, does not set forth
any date for the work she did or observed.
19
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