Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1995-02-13
1998-04-07
Jacobson, Dian C.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
424 9463, 435212, 435226, 4352402, 4352523, 536 232, A61K 3849, C12N 948, C12N 964, C12N 1555
Patent
active
057361355
ABSTRACT:
A screening method for the selection of mutagenized proteins that are normally secreted by cells is described. The method includes the development of a cloning vector for the expression of secretory proteins as fusion proteins on the cell surface of transfected mammalian cells. The secreted protein is displayed on the cell surface by fusion with the glycophospholipid membrane anchor of decay accelerating factor (DAF). Tissue-type plasminogen activator (t-PA), which is normally secreted, is used as a model protein. PCR mutagenesis is used to generate random mutations within the Kringle 1 (K1) domain of t-PA. Fluorescence activated cell sorting (FACS) is employed to screen for t-PA mutants possessing a loss of an epitope to a specific Mab, whose nonlinear binding domains overlap with the t-PA clearance receptor contact regions novel t-PA mutants designated N115S, N1425S, and K159R were discovered by this method.
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Goeddel David V.
Leung David W. H.
Rice Glenn C.
Dreger Ginger R.
Genentech Inc.
Jacobson Dian C.
Winter Daryl B.
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