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Protein Engineering, Vol. 13, No. 5, 377-384, May 2000
© 2000 Oxford University Press

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

Birgit Morawski, Zhanglin Lin1, Pat Cirino, Hyun Joo, Geethani Bandara and Frances H. Arnold2

Division of Chemistry and Chemical Engineering 210–41, California Institute of Technology, Pasadena, CA 91125, USA

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] (260 units/l/OD600). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD600 in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.


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