Method for making variant secreted proteins with altered properties
US5736135A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Feb 13, 1995 |
| Grant date | Apr 7, 1998 |
| Priority date | — |
| Expiry date | Feb 13, 2015 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC07K2319/33
- WIPO fieldPharmaceuticals
- WIPO sectorChemistry
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.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.