Recombinant DNA cloning vehicle

• US 4,356,270 A
• Filed: 11/05/1979
• Issued: 10/26/1982
• Est. Priority Date: 11/08/1977
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Itakura, Keiichi
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US06/091,334
• Time in Patent Office: 1,086 Days
• Field of Search: 435/172, 435/317, 435/68, 435/70, 435/71
• US Class Current: 435/320.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: Y10S 930/16 Somatostatin; related peptides

Abstract

The Specification discloses:

1. Recombinant microbial cloning vehicles comprising heterologous DNA coding for the expression of mammalian hormone (e.g., somatostatin) and other polypeptides, including plasmids suited for the transformation of bacterial hosts. The latter incorporate a regulon homologous to the host in its untransformed state, in reading phase with the structural gene for the heterologous DNA;

2. Cloning vehicles coding for the microbial expression of a protein variously comprising (a) a polypeptide hapten and additional protein sufficient in size to confer immunogenicity on the product of expression, which may find use in raising antibodies to the hapten for assay use or in the manufacture of vaccines; and (b) a desired polypeptide product and additional protein from which the desired product may be cleaved; and

3. Methods of preparing synthetic structural genes coding for the expression of mammalian polypeptides in microbial cloning systems.

Claims

1. A recombinant microbial cloning vehicle comprising a first restriction endonuclease recognition site, a structural gene coding for the expression of the amino acid sequence of a mammalian polypeptide, and a second restriction endonuclease site, at least a majority of the codons of said structural gene being codons preferred for the expression of microbial genomes.

2. A bacterial plasmid according to claim 1, the polypeptide being a hormone.

3. A plasmid according to claim 2 wherein said hormone is somatostatin.

4. A plasmid according to claim 3 wherein the codons for somatostatin are selected as follows:

space="preserve" listing-type="tabular">______________________________________ Codon from the group amino acid codon amino acid consisting of;

______________________________________ gly;

GGT ala;

GCT and GCG cys TGT asn;

AAT and AAC lys AAG phe;

TTC and TTT trp TGG thr;

ACT and ACG ser;

TCC and TCG. ______________________________________

5. A plasmid according to claim 3 comprising the base pair sequence:

##EQU1##

6. A plasmid according to claim 2 wherein the polypeptide encoded by said structural gene is the A chain of human insulin.

7. A plasmid according to claim 6 comprising the base pair sequence:

##EQU2##

8. A plasmid according to claim 2 wherein the polypeptide encoded by the structural gene is the B chain of human insulin.

9. A plasmid according to claim 8 comprising the base pair sequence:

##EQU3##