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Applied and Environmental Microbiology, February 2003, p. 1108-1113, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1108-1113.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

José Hanquier,1 Yannick Sorlet,2 Dominique Desplancq,2 Laurence Baroche,2 Marc Ebtinger,3 Jean-François Lefèvre,2 Franc Pattus,2 Charles L. Hershberger,1 and Alain A. Vertès3*

Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly & Co., Indianapolis, Indiana 46285 ,1 GIB-ESBS, F-67400 Illkirch,2 Lilly France, 67642 Fegersheim Cedex, France3

Received 4 September 2002/ Accepted 8 November 2002

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.

* Corresponding author. Present address: Pfizer Global Research & Development, 3-9 Rue de la Loge, BP 100, F-94265, Fresnes Cedex, France. Phone: 01 40 96 74 00. Fax: 01 46 68 16 44. E-mail: alain.vertes{at}pfizer.com.

Applied and Environmental Microbiology, February 2003, p. 1108-1113, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1108-1113.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Vertes, A. A., Inui, M., Yukawa, H. (2005). Manipulating Corynebacteria, from Individual Genes to Chromosomes. Appl. Environ. Microbiol. 71: 7633-7642 [Full Text]  


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