title: High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).
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. coli, when compared to His-tag alone.
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, 13566-590 São Carlos, SP, Brazil.
authors.forename: Cesar M
authors.initials: CM
authors.lastname: Camilo
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, 13566-590 São Carlos, SP, Brazil. Electronic address: ipolikarpov@ifsc.usp.br.
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D004259:DNA-Directed DNA Polymerase; D006026:Glycoside Hydrolases
keywords: Glycoside hydrolases; High-throughput; LIC; Ligation-Independent Cloning; Solubility screening; Thioredoxin