A single mutation in the activation site of bovine trypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris.
We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.
Hanquier
José
J
Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly & Co., Indianapolis, Indiana 46285, USA.
Sorlet
Yannick
Y
Desplancq
Dominique
D
Baroche
Laurence
L
Ebtinger
Marc
M
Lefèvre
Jean-François
JF
Pattus
Franc
F
Hershberger
Charles L
CL
Vertès
Alain A
AA
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003470:Culture Media; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014362:Trypsinogen / Q000235:genetics* / Q000378:metabolism
D003470:Culture Media; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D014362:Trypsinogen; D014357:Trypsin