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

• Glycoside hydrolases were submitted to a high throughput cloning, expression and purification protocol.
• 130 genes were cloned into two bacterial expression vectors providing N-6xHis-TEV and N-6xHis-TRX-TEV fusion tags.
• 56.2% of the targets rendered soluble protein in at least one of the expression vectors.
• Thioredoxin was a better fusion tag for glycoside hydrolase expression in Escherichia coli when compared to 6xHis tag alone.

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.