Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Shubhada Godbole; Stephen R. Decker; Rafael A. Nieves; William S. Adney; Todd B. Vinzant; John O. Baker; Steven R. Thomas; Michael E. Himmel

doi:10.1021/bp9901116

Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Shubhada Godbole, Stephen R. Decker, Rafael A. Nieves, William S. Adney, Todd B. Vinzant, John O. Baker, Steven R. Thomas, Michael E. Himmel

National Renewable Energy Laboratory

Research output: Contribution to journal › Article › peer-review

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Abstract

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.

Original language	American English
Pages (from-to)	828-833
Number of pages	6
Journal	Biotechnology Progress
Volume	15
Issue number	5
DOIs	https://doi.org/10.1021/bp9901116 https://doi.org/10.1021/bp9901116
State	Published - 1999

NREL Publication Number

NREL/JA-580-27873

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title = "Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris",

abstract = "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.",

author = "Shubhada Godbole and Decker, {Stephen R.} and Nieves, {Rafael A.} and Adney, {William S.} and Vinzant, {Todd B.} and Baker, {John O.} and Thomas, {Steven R.} and Himmel, {Michael E.}",

year = "1999",

doi = "10.1021/bp9901116",

language = "American English",

volume = "15",

pages = "828--833",

journal = "Biotechnology Progress",

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T1 - Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

AU - Godbole, Shubhada

AU - Decker, Stephen R.

AU - Nieves, Rafael A.

AU - Adney, William S.

AU - Vinzant, Todd B.

AU - Baker, John O.

AU - Thomas, Steven R.

AU - Himmel, Michael E.

PY - 1999

Y1 - 1999

N2 - 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.

AB - 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.

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U2 - 10.1021/bp9901116

DO - 10.1021/bp9901116

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AN - SCOPUS:0033200293

SN - 8756-7938

VL - 15

SP - 828

EP - 833

JO - Biotechnology Progress

JF - Biotechnology Progress

IS - 5

ER -

Cloning and Expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Abstract

NREL Publication Number

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