Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris.
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/OD(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) 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.
Morawski
B
B
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Lin
Z
Z
Cirino
P
P
Joo
H
H
Bandara
G
G
Arnold
F H
FH
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D006735:Horseradish Peroxidase