Expression of synthetic thaumatin genes in yeast.
Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.
Lee
J H
JH
INGENE, Inc., Santa Monica, California 90404.
Weickmann
J L
JL
Koduri
R K
RK
Ghosh-Dastidar
P
P
Saito
K
K
Blair
L C
LC
Date
T
T
Lai
J S
JS
Hollenberg
S M
SM
Kendall
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013549:Sweetening Agents
D010940:Plant Proteins; D013549:Sweetening Agents; C003427:thaumatin protein, plant; D010735:Phosphoglycerate Kinase