Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris.
Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels. Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme. More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.
Godbole
S
S
Biotechnology Center for Fuels and Chemicals, National Renewable Energy Laboratory, Golden, Colorado 80401, USA.
Decker
S R
SR
Nieves
R A
RA
Adney
W S
WS
Vinzant
T B
TB
Baker
J O
JO
Thomas
S R
SR
Himmel
M E
ME
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020539:Sequence Analysis, Protein; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase