US5223408A
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Method for making variant secreted proteins with altered properties
Assignee: Genentech Inc
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Filing: 1991-07-11
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Granted: 1993-06-29
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Expires: 2011-07-11
Abstract
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.
Inventors
- 👤 David V. Goeddel
- 👤 Glenn C. Rice
- 👤 David W. H. Leung
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 07:46
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/1034; C12N15/1037; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; C12N9/6459; C12Y; C12Y304/00; C12Y304/21; C12Y304/21069; A; A61; A61K; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/033; C07K2319/33
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
211 words
A method for making a variant secretory protein retaining at least one desired binding property and eliminating at least one undesired binding property of the wild-type secretory protein comprising: (a) mutating the DNA encoding the selected wild-type protein thereby creating a library of related variant DNA molecules; (b) inserting each DNA molecule created in step (a) into an expression vector, wherein the vector comprises DNA encoding a transmembrane anchor domain thereby creating a library of vectors; (c) transfecting eukaryotic cells with the vectors of step (b); (d) culturing the cells of step (c) under conditions inducing the expression of the DNA to produce a chimeric fusion protein immobilized on the cell membrane; (e) contacting the cultured cells of step (d) with first and second reporter molecules capable of binding to different epitopes on the wild-type protein, each reporter molecule containing a fluorophore different from the fluorophore of the other reporter molecule, with said contacting taking place under conditions for which at least a portion of the cultured cells bind to the first or second reporter molecules; (f) sorting the contacted cells based on a desired binding pattern with the first or second reporter molecules; and (g) obtaining the variant proteins having the desired binding pattern from the sorted cells from (f).
Claim 2
Independent Claim
211 words
A method for making a variant secretory protein retaining at least one desired binding property and eliminating at least one undesired binding property of the wild-type secretory protein comprising: (a) mutating the DNA encoding the selected wild-type protein thereby creating a library of related variant DNA molecules; (b) inserting each DNA molecule created in step (a) into an expression vector, wherein the vector comprises DNA encoding a transmembrane anchor domain thereby creating a library of vectors; (c) transfecting eukaryotic cells with the vectors of step (b); (d) culturing the cells of step (c) under conditions inducing the expression of the DNA to produce a chimeric fusion protein immobilized on the cell membrane; (e) contacting the cultured cells of step (d) with first and second reporter molecules capable of binding to different epitopes on the wild-type protein, each reporter molecule containing a fluorophore different from the fluorophore of the other reporter molecule, with said contacting taking place under conditions for which at least a portion of the cultured cells bind to the first or second reporter molecules; (f) sorting the contacted cells based on a desired binding pattern with the first or second reporter molecules; and (g) obtaining the variant proteins having the desired binding pattern from the sorted cells from (f).
Claim 3
Dependent Claim
17 words
The method of claim 1 wherein step (g) comprises isolating the variant protein from the sorted cells.
Claim 4
Dependent Claim
28 words
The method of claim 1 wherein step (g) comprises isolating the variant DNA molecule and producing the variant protein from the isolated variant DNA molecule by recombinant methods.
Claim 5
Dependent Claim
56 words
The method of claim 1 wherein step (g) comprises sequencing the variant DNA molecule, determining the codons encoding amino acids in the variant protein that produce the desired binding pattern, altering those codons in a DNA molecule encoding the wild-type protein, and producing the variant protein from the DNA containing the altered codons by recombinant methods.
Claim 6
Dependent Claim
29 words
The method of claim 1 wherein the first and second reporter molecules comprise a detectable marker conjugated to a molecule selected from the group; antibodies, ligands, and soluble receptors.
Claim 7
Dependent Claim
27 words
The method of claim 5 wherein the first and second reporter molecules comprise a first and a second monoclonal antibody (Mab) each conjugated to a different fluorophore.
Claim 8
Dependent Claim
15 words
The method of claim 6 wherein the sorting step comprises fluoresence activated cell sorting (FACS).
Claim 9
Dependent Claim
24 words
The method of claim 1 wherein the DNA encoding the transmembrane anchor domain encodes the membrane glycophospholipid anchor sequence of decay acceleration factor (DAF).
Claim 10
Dependent Claim
176 words
The method of claim 8 wherein the selected wild-type protein is selected from the group; growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β, platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferon alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, superoxide dismutase; viral antigens, HIV envelope proteins GP120 and GP140, immuno globulins, and fragments of the above-listed proteins.
Claim 11
Dependent Claim
12 words
The method of claim 9 wherein the protein is a human protein.
Claim 12
Dependent Claim
13 words
The method of claim 1 wherein the eukaryotic cell is a mammalian cell.
Claim 13
Dependent Claim
33 words
The method of claim 1 wherein the desired binding pattern comprises binding of the cells with the first reporter molecule and the absence of binding of the cells with the second reporter molecule.
References Cited (Backward Citations)
No citation records indexed.
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