Ruth Larragy

Investigation into dissolved neutral sugars and their microbial conversion in natural and artificially produced dissolved organic matter using ion chromatography with pulsed amperometric detection and reversed-phase liquid chromatography-high resolution mass spectrometry

Ion-exchange chromatography with pulsed amperometric detection (IEC-PAD) was employed to investigate dissolved neutral sugars and their microbial conversion in both artificially prepared dissolved organic matter (ADOM), and naturally occurring dissolved organic matter (DOM) obtained from seawater and freshwater sources. The analysis of ADOM and naturally occurring DOM samples using IEC-PAD resulted in chromatograms suggesting very similar composition, each characterised by three early eluting peaks, the latter of which being a broad co-elution of multiple compounds. For naturally occurring DOM, several sugars, including arabinose, glucose, galactose, xylose and ribose, could also be identified. The three distinctive peaks obtained from IEC-PAD of ADOM were collected and further analysed by means of reversed-phase high performance liquid chromatography-high resolution mass spectrometry (RPLC-HRMS), the latter showing that glucose was totally consumed during microbial production of ADOM and potentially transformed into higher molecular weight materials and CO2.

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

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.