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

Author(s)

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

Affiliation

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

• Document type: Journal article
• Source: Protein Expression and Purification; v. 99, p. 35-42, JUL 2014.
• Web of Science Citations: 21

Abstract

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)

• FAPESP's process: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Grantee: Igor Polikarpov
• Support type: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 10/08680-2 - Molecular aspectos of lignocellulosic biomass degradation: dynamics of enzymes and plant cell wall nanoarchitecture
• Grantee: Rodrigo Leandro Silveira
• Support type: Scholarships in Brazil - Doctorate
• FAPESP's process: 09/54035-4 - Facility for advanced studies of biosystems and nanostructured materials
• Grantee: Igor Polikarpov
• Support type: Multi-user Equipment Program
• FAPESP's process: 09/11536-3 - Biochemical and structural studies of the cellulolytic system of Trichoderma reesei and Phanerochaete chrysosporium, applied to improve the efficiency biomass degradation
• Grantee: Cesar Moises Camilo
• Support type: Scholarships in Brazil - Post-Doctorate