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. The authors conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.
- Research Organization:
- National Renewable Energy Lab., Golden, CO (US)
- OSTI ID:
- 20001020
- Journal Information:
- Biotechnology Progress, Journal Name: Biotechnology Progress Journal Issue: 5 Vol. 15; ISSN 8756-7938; ISSN BIPRET
- Country of Publication:
- United States
- Language:
- English
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