Generated: April 25, 2017
|Title:||Method of constructing a replicable cloning vehicle having quasi-synthetic genes|
|Abstract:||Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.|
|Inventor(s):||Goeddel; David V. (Burlingame, CA), Heyneker; Herbert L. (Burlingame, CA)|
|Assignee:||Genentech, Inc. (South San Francisco, CA)|
|Filing Date:||Jul 05, 1979|
|Claims:||1. In the method of constructing a replicable cloning vehicle capable, in a microbial organism, of expressing a particular polypeptide of known amino acid sequence wherein a gene coding for the polypeptide is inserted into a cloning vehicle and placed under the control of an expression promoter, the improvement which comprises: |
(a) obtaining by reverse transcription from messenger RNA a first gene fragment for an expression product other than said polypeptide, which fragment comprises at least a portion of the coding sequence for said polypeptide;
(b) where the first fragment comprises protein-encoding codons for amino acid sequences other than those contained in said polypeptide, eliminating the same while retaining at least a substantial portion of said coding sequence, the resulting fragment nevertheless coding for an expression product other than said polypeptide;
the product of step (a) or, where required, step (b) being a fragment encoding less than all of the amino acid sequence of said polypeptide;
(c) providing by organic synthesis one or more synthetic non-reverse transcript-gene fragments encoding the remainder of the amino acid sequence of said polypeptide, at least one of said fragments coding for the amino-terminal portion of the polypeptide; and
(d) deploying the synthetic gene fragment(s) of step (c) and that produced in step (a) or (b), as the case may be, in a replicable cloning vehicle in proper reading phase relative to one another and under the control of an expression promoter;
whereby a replicable cloning vehicle capable of expressing the amino acid sequence of said polypeptide is formed.
2. The method of claim 1 wherein the cloning vehicle of step (d) is a bacterial plasmid.
3. The method of claim 2 wherein the synthetic fragment encoding the amino-terminal portion of the polypeptide additionally codes for expression of a specifically cleavable amino acid sequence, and wherein the fragments are deployed downstream from and in reading phase with expressed protein-encoding condons, whereby the conjugated plasmid expression product may be specifically cleaved to yield the polypeptide.
4. The method of claim 2 wherein the amino acid sequence of the polypeptide is expressable unaccompanied by extraneous protein.
5. The method of claim 4 wherein the fragment of step (a) comprises at least a majority of the coding sequence for said polypeptide.
6. The method of claim 2 wherein a synthetic fragment and an mRNA transcript fragment are ligated to one another before their deployment in the cloning vehicle, and wherein the opposite ends of the fragment and of the transcript are variously single stranded or blunt so as to ensure ligation of the two fragments in the proper order for expression of said polypeptide.
7. The method of claim 5 wherein the polypeptide is human growth hormone, and wherein the first fragment comprises protein-encoding codons for amino acid sequences other than those in human growth hormone, and wherein elimination step (b) yields the Hae III restriction enzyme fragment of the first fragment.
8. The method of claim 7 wherein step (b) includes digestion of the Hae III fragment with a different restriction enzyme, cleaving away codons for untransulated messenger RNA and simultaneously providing a single-stranded terminus at one end of the resulting fragment.
9. The method of claim 8 wherein the second restriction enzyme is Xma I.
10. A method according to claim 1 wherein the polypeptide is human growth hormone and wherein the codons for amino acids 1-24 thereof are essentially as depicted in FIG. 1.
11. A method according to claim 4 wherein the polypeptide is human growth hormone and wherein the codons for amino acids 1-24 thereof are essentially as depicted in FIG. 1.
12. A method according to claim 7 wherein the codons for amino acids 1-24 are essentially as depicted in FIG. 1.
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