Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant Trichoderma reesei strains devoid of major endoglucanase genes

Anu Koivula, Arja Lappalainen, Susanna Virtanen, Arja Mäntylä, Pirkko Suominen, Tuula Teeri

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.
Original languageEnglish
Pages (from-to)391-400
Number of pages10
JournalProtein Expression and Purification
Volume8
Issue number4
DOIs
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Cellulase
Genes
Catalytic Domain
Cellulases
Cyanogen Bromide
Affinity Chromatography
Cellulose
Immobilization
Culture Media
Chromatography
Proteins
Monoclonal Antibodies

Cite this

Koivula, Anu ; Lappalainen, Arja ; Virtanen, Susanna ; Mäntylä, Arja ; Suominen, Pirkko ; Teeri, Tuula. / Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant Trichoderma reesei strains devoid of major endoglucanase genes. In: Protein Expression and Purification. 1996 ; Vol. 8, No. 4. pp. 391-400.
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abstract = "Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.",
author = "Anu Koivula and Arja Lappalainen and Susanna Virtanen and Arja M{\"a}ntyl{\"a} and Pirkko Suominen and Tuula Teeri",
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Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant Trichoderma reesei strains devoid of major endoglucanase genes. / Koivula, Anu; Lappalainen, Arja; Virtanen, Susanna; Mäntylä, Arja; Suominen, Pirkko; Teeri, Tuula.

In: Protein Expression and Purification, Vol. 8, No. 4, 1996, p. 391-400.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant Trichoderma reesei strains devoid of major endoglucanase genes

AU - Koivula, Anu

AU - Lappalainen, Arja

AU - Virtanen, Susanna

AU - Mäntylä, Arja

AU - Suominen, Pirkko

AU - Teeri, Tuula

N1 - Project code: BEL4319

PY - 1996

Y1 - 1996

N2 - Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.

AB - Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.

U2 - 10.1006/prep.1996.0116

DO - 10.1006/prep.1996.0116

M3 - Article

VL - 8

SP - 391

EP - 400

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

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