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US 5,536,637 AGranted
Method of screening for cDNA encoding novel secreted mammalian proteins in yeast
AssigneeGenetics Institute, Inc.
InventorKenneth Jacobs
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Abstract
A novel method of screening for novel secreted mammalian proteins is described in which mammalian secretory leader sequences are detected using the yeast invertase gene as a reporter system.
Claims1
A method of screening for a cDNA encoding a novel secreted mammalian protein which comprises:
- a) constructing a cDNA library from mammalian cellular RNA;
- b) ligating said cDNA library to a DNA encoding a nonsecreted yeast invertase;
- c) transforming the ligated DNA into E. coli;
- d) isolating DNA containing mammalian cDNA ligated to the DNA encoding the nonsecreted yeast invertase from the transformed E. coli of step c);
- e) transforming the DNA of step d) into a yeast cell which does not contain an invertase gene;
- f) selecting yeast cells capable of growth on sucrose or raffinose;
- g) purifying DNA from the yeast cells of step f);
- h) analyzing the DNA obtained from step g) to determine its sequence and to determine whether it contains a novel sequence;
- i) screening a second cDNA library to detect a full-length cDNA which contains the novel sequence of step h);
- j) isolating the full-length cDNA of step i) wherein the isolated cDNA encodes a putative secreted mammalian protein.
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