Abstract
A method is provided for synthesizing within a bacterial host, and secreting through the membrane of the host, a selected mature protein or polypeptide. The method involves:
Filing date: Apr 11, 1980
Issue date: Jul 6, 1982
Inventors: Walter Gilbert, Karen Talmadge
Assignee: President and Fellows of Harvard College
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What is claimed is:
1. A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises:
- (a) cleaving a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host, to form a cleavage site after a promoter of either (1) a bacterial or phage gene within the cloning vehicle or (2) a DNA fragment of the bacterial or phage gene;
- (b) forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide;
- (c) transforming the bacterial host with the cloning vehicle; and then
- (d) culturing the transformed bacterial host to secrete the selected protein or polypeptide.
2. The method of claim 1 wherein the bacterial or phage gene or the DNA fragment thereof codes for a normally secreted protein or polypeptide and the cleavage site is before, within or no more than approximately 60 nucleotides after the translational start signal of the bacterial or phage gene or the DNA fragment thereof.
3. The method of claim 1 wherein the bacterial or phage gene or the DNA fragment thereof codes for a normally non-secreted protein or polypeptide and the cleavage site is before, within or no more than approximately 40 nucleotides after the translational start signal of the bacterial or phage gene or the DNA fragment thereof.
4. The method of claims 2 or 3 wherein the cleavage site is within or no more than approximately 40 nucleotides after the translational start signal of the bacteria or phage gene or the DNA fragment thereof.
5. The method of claim 1 wherein a DNA fragment of the E. coli penicillinase gene is cleaved.
6. The method of claim 5 wherein the cloning vehicle is derived from the plasmid pBR322.
7. The method of claim 1 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
8. A cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in a bacterial host and comprising in order:
- a promoter of a bacterial or phage gene;
- a translation start signal; and
- a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame define by the translational start signal.
9. The cloning vehicle of claim 8 which further includes a ribosome binding site between the promoter and the translational start signal.
10. The cloning vehicle of claim 9 wherein the promoter is the promoter of the E. coli penicillinase gene.
11. The cloning vehicle of claim 9 which further includes no more than approximately 40 nucleotides of the bacterial or phage gene after the translational start signal and before the non-bacterial DNA fragment.
12. The cloning vehicle of claim 9 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
13. A bacterial host transformed with a cloning vehicle, the cloning vehicle comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order:
- a promoter of a bacterial or phage gene;
- a translational start signal; and
- a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
14. The host of claim 13 which further includes a ribosome binding site between the promoter and the translational start signal.
15. The host of claim 14 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
16. A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises culturing the bacterial host; the bacterial host being transformed with a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order:
- a promoter of a bacterial or phage gene;
- a translational start signal; and
- a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
17. The method of claim 16 wherein the cloning vehicle further includes a ribosome binding site between the promoter and the translational start signal.
18. The method of claim 17 wherein the promoter is the promoter of the E. coli penicillinase gene.
19. The method of claim 17 wherein the cloning vehicle further includes no more than approximately 40 nucleotides of the bacterial or phage gene after the translational start signal and before the non-bacterial DNA fragment.