Enhanced production and purification of a natural high intensity sweetener
AssigneeTate & Lyle Technology Limited
InventorsAlfred Carlson, Richard W. Armentrout, Timothy Peter Ellis
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Abstract
Recombinant P. pastoris producing natural sweet proteins and methods for engineering these recombinant yeast are described. Methods for enhancing foreign protein production in yeast fermentation and improved methods for purification of foreign proteins produced in yeast fermentation are presented.
Claims26
An expression vector for the production of a brazzein protein by a yeast cell comprising at least two cassettes, each cassette comprising a promoter sequence, a secretion signal sequence, a brazzein protein reading frame sequence, and a termination sequence, wherein the at least two cassettes are present in an expression vector comprising an integration sequence for integration of the cassettes into the yeast genome at a specific site.
A recombinant P. pastoris cell comprising at least two copies of a gene construct comprising a pGAP promoter sequence, a secretion signal sequence, a brazzein Type II or Type III protein reading frame sequence, and a termination sequence, wherein the copies of the gene construct are present at a preselected integration site within the P. pastoris genome.
A method for enhancing production of a foreign protein in recombinant Pichia comprising the steps of a) initiating a culture of the recombinant Pichia, under aerobic conditions, b) reducing oxygen levels in the culture to less than 5%, and c) maintaining oxygen levels in the culture at less than 5%.
A method for purifying a protein from a yeast fermentation culture comprising the ordered steps of a) adjusting the pH of the fermentation medium above or below the isoelectric point of the protein to be purified;
- b) removing the cells from the pH adjusted fermentation medium;
- c) subjecting the cell-free fermentation medium to ion-exchange chromatography and eluting the protein as a solution;
- d) passing the eluted protein solution through a first membrane to remove unwanted high molecular weight solutes;
- and e) concentrating and diafiltering the protein permeate on a second membrane to remove low molecular weight solutes.
Dependencies are inferred from the claim text.