Generated: April 27, 2017
|Title:||Human growth hormone|
|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:||Jun 01, 1995|
|Claims:||1. A method of producing a polypeptide of known amino acid sequence by constructing a replicable cloning vehicle containing DNA encoding said polypeptide, said cloning vehicle contained within a microbial organism, wherein said DNA is under the control of an expression promotor contained in said replicable cloning vehicle, comprising: |
a) obtaining by reverse transcription from messenger RNA a first DNA fragment for an expression product other than said polypeptide, which fragment comprises at least a substantial 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 DNA 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 DNA 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; and
e) expressing said DNA to produce said polypeptide.
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