High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC)

Autor(es)

Camilo, Cesar M. ^[1] ; Polikarpov, Igor ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Fis Sao Carlos, Dept Fis & Informat, BR-13566590 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 99, p. 35-42, JUL 2014.
• Citações Web of Science: 23

Resumo

Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coil, when compared to His-tag alone. (C) 2014 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Beneficiário: Igor Polikarpov
• Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Temático
• Processo FAPESP: 10/08680-2 - Aspectos moleculares da degradação de biomassa lignocelulósica: dinâmica de enzimas e nanoarquitetura de paredes celulares de plantas
• Beneficiário: Rodrigo Leandro Silveira
• Linha de fomento: Bolsas no Brasil - Doutorado
• Processo FAPESP: 09/54035-4 - EMU: facility para estudos avançados de materiais nanoestruturados e biossistemas / FAMA
• Beneficiário: Igor Polikarpov
• Linha de fomento: Auxílio à Pesquisa - Programa Equipamentos Multiusuários
• Processo FAPESP: 09/11536-3 - Estudos bioquímicos e estruturais dos sistemas celulolíticos de Trichoderma reesei e Phanerochaete chrysosporium aplicados à melhoria da eficiência de degradação de biomassa
• Beneficiário: Cesar Moises Camilo
• Linha de fomento: Bolsas no Brasil - Pós-Doutorado