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 hepten 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.
A recombinant microbial cloning vehicle comprising a regulon, a structural gene coding for the amino acid sequence of a desired heterologous polypeptide and one or more termination codon(s) wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that a precursor protein comprising both the amino acid sequence of the desired heterologous polypeptide and that of additional protein results from expression, the additional protein comprising a selective cleavage site adjacent the amino acid sequence of the desired heterologous polypeptide.
Claim 2
Dependent Claim
95 words
A recombinant microbial cloning vehicle comprising a regulon, a structural gene coding for the amino acid sequence of a desired heterologous polypeptide and one or more termination codon(s) wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that a precursor protein comprising both the amino acid sequence of the desired heterologous polypeptide and that of additional protein results from expression, the additional protein comprising a selective cleavage site adjacent the amino acid sequence of the desired heterologous polypeptide.
Claim 3
Dependent Claim
16 words
The vehicle of claim 1 wherein the desired heterologous polypeptide contains no similar selective cleavage site.
Claim 4
Dependent Claim
24 words
A bacterial plasmid according to claim 2 wherein the recited elements are sequenced as follows: (regulon)-(codons for additional protein)-(codons for desired heterologous polypeptide)-(termination codon(s)).
Claim 5
Dependent Claim
12 words
A plasmid according to claim 3 wherein the cleavage site is methionine.
Claim 6
Dependent Claim
13 words
A plasmid according to claim 4 wherein the desired heterologous polypeptide is somatostatin.