Damien Keogh1, Roisin Thompson2, Ruth Larragy2, Kenneth McMahon1, Michael O'Connell1, Brendan O'Connor2, Paul Clarke3. 1. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. 2. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; Irish Separation Science Cluster (ISSC), National Centre for Sensor Research (NCSR), Dublin City University, Glasnevin, Dublin 9, Ireland. 3. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; Irish Separation Science Cluster (ISSC), National Centre for Sensor Research (NCSR), Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address: Paul.Clarke3@gmail.com.
Abstract
BACKGROUND: Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs. METHOD: Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties. RESULTS: This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated. CONCLUSIONS: The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties. GENERAL SIGNIFICANCE: The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.
BACKGROUND: Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs. METHOD: Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties. RESULTS: This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated. CONCLUSIONS: The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties. GENERAL SIGNIFICANCE: The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.
Authors: Elizabeth Edwards; Maria Livanos; Anja Krueger; Anne Dell; Stuart M Haslam; C Mark Smales; Daniel G Bracewell Journal: Biotechnol Bioeng Date: 2022-02-28 Impact factor: 4.395