High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.
Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.
Cousens
L S
LS
Chiron Corporation, Emeryville, CA 94608.
Shuster
J R
JR
Gallegos
C
C
Ku
L L
LL
Stempien
M M
MM
Urdea
M S
MS
Sanchez-Pescador
R
R
Taylor
A
A
Tekamp-Olson
P
P
D002874:Chromosome Mapping; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D008854:Microscopy, Electron; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D011384:Proinsulin