Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa
| Autor: | Roisin Thompson, Damien Keogh, Michael O'Connell, Ruth Larragy, Paul M. Clarke, Brendan O'Connor, Kenneth Mcmahon |
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| Rok vydání: | 2014 |
| Předmět: |
Glycan
Carbohydrates Biophysics Mutagenesis (molecular biology technique) Biology 01 natural sciences Biochemistry law.invention 03 medical and health sciences law Lectins Binding site Adhesins Bacterial Molecular Biology 030304 developmental biology Galectin chemistry.chemical_classification 0303 health sciences Glycobiology 010401 analytical chemistry Lectin Recombinant Proteins 0104 chemical sciences chemistry Pseudomonas aeruginosa Mutagenesis Site-Directed Recombinant DNA biology.protein Glycoprotein |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1840:2091-2104 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/j.bbagen.2014.01.020 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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