Method for identification and isolation of DNA encoding a desired protein

• US 4,675,285 A
• Filed: 09/19/1984
• Issued: 06/23/1987
• Est. Priority Date: 09/19/1984
• Status: Expired due to Term

• Current Assignee: Genetics Institute LLC (Pfizer Inc.)
• Original Assignee: Genetics Institute LLC (Pfizer Inc.)
• Inventors: Kaufman, Randal J., Clark, Steven C., Wong, Gordon G.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Jay, Jeremy
• Application Number:
  US06/652,316
• Time in Patent Office: 1,007 Days
• Field of Search: 435/6, 435/68, 435/91, 435/172.3, 435/240, 435/241, 435/317, 435/948, 435/255, 435/253, 935/2, 935/11, 935/19, 935/21, 935/28, 935/29, 935/42, 935/43, 935/47, 935/48, 935/56, 935/60, 935/69, 935/70, 935/72, 935/78, 935/79
• US Class Current: 435/6.16
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/85 for animal cells

Abstract

A method for identifying and isolating clones containing DNA coding for a desired protein is described. DNA prepared from a cell that expresses the desired protein is inserted into an isolation expression vector having means for replication (as a means of producing DNA) and a suitable promoter for expression of said DNA in a predetermined mammalian host cell as well as means for replication in a bacterial cell. The transient expression vector is then inserted into a bacterial cell for replication of the DNA. Pools of DNA, prepared from a predetermined number of bacterial clones so that the nucleic acids (DNA and RNA) is substantially free of other bacterial contaminants are transfected or microinjected into mammalian host cells and conditioned medium from growing such cells is tested for the presence of the desired protein. Positive pools are selected and the clones used to make the pool are screened to identify and isolate the clone containing the desired DNA.

Claims

1. A method 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 said cDNA library into isolation expression vectors, said vectors having regulatory sequences capable of directing replication in bacterial cells and a promoter that directs replication and expression of said cDNA in predetermined mammalian cells;

(c) inserting said vectors into a bacterial cells and culturing said bacterial cell to produce clones;

(d) collecting pools of a predetermined number of colonies of said bacterial clones;

(e) preparing DNA from each said pool by substantially removing bacterial contaminants therefrom, which inhibit expression of said DNA in mammalian cells;

(f) transfecting the DNA from each pool into said mammalian cells;

(g) culturing said transfected mammalian cells to produce mammalian cell cultures and testing media from each of said cell cultures for expression of the activity of the desired protein;

(h) selecting bacterial pools which correspond to the mammalian cell cultures expressing said activity;

(i) repeating steps (e) through (h) 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 identified in step (i).

2. The method according to claim 1, wherein said mammalian cells are COS cells.

3. The method according to claim 1, wherein said protein is a secreted protein.

4. The method according to claim 1, wherein said protein has a molecular weight up to about 100,000 daltons.

5. The method according to claim 1, wherein said isolation expression vectors are selected from the group consisting of high copy number vectors and vectors amplifiable with chloramphenicol.

6. The method according to claim 1, wherein said isolation expression vector is p91023(B).

7. The method according to claim 1, wherein said cDNA encoding said protein contains about 4000 nucleotides.

8. The method according to claim 7, wherein said cDNA encoding said protein contains about 2000 nucleotides.