Method for identification and isolation of DNA encoding a desired protein

Patent · Tue Jun 23 04:00:00 EDT 1987

OSTI ID:6076112

Clark, S C; Kaufman, R J; Wong, G G

A method is described for identifying a cDNA sequence encoding a desired protein, comprising the steps of: (a) preparing a cDNA library from a cell line that expresses the desired protein; (b) inserting the cDNA library into isolation expression vectors, the vectors having regulatory sequences capable of directing replication in bacterial cells and a promoter that directs replication and expression of the cDNA in predetermined mammalian cells; (c) inserting the vectors into a bacterial cells and culturing the bacterial cell to produce clones; (d) collecting pools of a predetermined number of colonies of the bacterial clones; (e) preparing DNA from each pool by substantially removing bacterial contaminants, which inhibit expression of DNA in mammalina cells; (f) transfecting the DNA from each pool into the mammalian cells; (g) culturing transfected mammalina cells to produce mammalian cell cultures and testing media from each of cell cultures for expression of the activity of the desired protein; (h) selecting bacterial pools which correspond to the mammalian cell cultures expressing the activity; (i) repeating until a colony of the bacterial cells containing the cDNA that expresses the desired protein when introduced into mammalian cells is identified; and (j) excising the cDNA from the bacterial colony.

• Assignee: Genetics Institute, Inc., Cambridge, MA
• Patent Number(s): US 4675285
• OSTI ID: 6076112
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
BACTERIA
CELL CONSTITUENTS
CELL CULTURES
CLONE CELLS
CLONING
COLONY FORMATION
DESIGN
DNA SEQUENCING
DNA-CLONING
GENE OPERONS
GENE REGULATION
GENETIC ENGINEERING
LIBRARIES
MICROORGANISMS
ORGANIC COMPOUNDS
PLASMIDS
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS