Genentech, Inc.

University of California

Dennis G Kleid

Herbert L Heyneker

Daniel G Yansura

Giuseppe F Miozzari

Debi P Nayak

Alan R Davis

Methods for the controllable expression in high yields of heterologous proteins under the control of a modified tryptophan promoter-operator system are disclosed. The system employed involves sequential resort to tryptophan repression and derepression, as well as deletion of an attenuator function native to the tryptophan operon. Means are also disclosed for effecting deletions at any given point within a gene fragment and for the ready identification of transformant bacteria via triple ligation procedures.

Bacterial polypeptide expression employing tryptophan promoter-operator

The present invention provides recombinant DNA vehicles which are suitable for the microbial expression of DNA encoding a heterologous polypeptide which comprises a portion of the trp operon having the promoter-operator and leader ribosome binding site, and a restriction site providing an insertion site for the DNA sequences encoding the heterologous polypeptide, wherein the restriction site is located 3' of the leader ribosome binding site as a substitute for the Taq I site of the trp promoter-operator and is selected from the group consisting of Xba I and Eco RI. Also provided are E. coli strains transformed with the above described recombinant DNA vehicles.

A method of altering double stranded DNA comprising converting double stranded DNA to single stranded DNA in a region surrounding a selected cleavage site, hybridizing a complimentary primer to the single stranded region formed in the previous step, and restoring enzymatically the second strand which had been eliminated.

Method of altering double-stranded DNA

Disclosed herein are methods and means for microbially expressing human influenza hemagglutinin proteins, useful in the preparation of vaccines, by recombination of DNA gene sequences coding for hemagglutinin protein with microbially operable promoter-operator DNA and transformation of microbial host therewith.

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San Mateo, CA, United States of America

Dennis G Kleid