•The cloning, expression, purification and characterisation of recombinant prokaryotic glycolytic enzymes

•The mutagenesis of prokaryotic glycolytic enzymes to generate novel recombinant carbohydrate binding proteins

•The characterisation of the binding profile of the novel recombinant carbohydrate binding protein

Clarke, Paul A.

Harrington, Amy

O'Connor, Brendan

Thompson, Roisin

English

•The cloning, expression, purification and characterisation of recombinant prokaryotic glycolytic enzymes\ud
•The mutagenesis of prokaryotic glycolytic enzymes to generate novel recombinant carbohydrate binding proteins\ud
•The characterisation of the binding profile of the novel recombinant carbohydrate binding protein

Name not available

GLYCOLYTIC ENZYMES - NOVEL CARBOHDRATE BINDING PROTEINS FOR GLYCOPROTEIN ANALYSISAmy Harrington1, Roisin Thompson1, Brendan O’Connor1, Paul Clarke 11. Irish Separation Science Cluster, Dublin City University, Dublin This research has been funded by Science Foundation Ireland under grant number  and the IRCSET Post Graduate Scholarship Scheme7. Project outputsAdvanced SilicaSupport  & Bonded Phase Materials(WP 1.2)Materials TechnologyAdvanced PlatformsAdvanced Polymer Substrates(WP 1.3)Nano-materials for Separation and Sensing(WP 1.4)Microfluidics(WP 2.1)Methods & Advanced Applications(WP 2.4)Instrumentation & Detection(WP 2.3)Bio-ligands & Purification(WP 1.1)Column Technology(WP 2.2)Aims:•The cloning, expression, purification and characterisation of recombinantprokaryotic glycolytic enzymes•The mutagenesis of prokaryotic glycolytic enzymes to generate novelrecombinant carbohydrate binding proteins•The characterisation of the binding profile of the novel recombinantcarbohydrate binding proteins6. Future Work• Enable detection of chb mutant in ELLAs – use of linker, longer His tagor biotinylation• Further characterization of binding once detection is enabled• Random mutagenesis to alter binding specificities/affinities00.050.10.150.20.250.30.350.40 20 40 60 80 100Absorbance 450nmConcentration chb mutant (ug/ml)Binding of GSLII to GlcNAc over a P. luminescens chb mutant concentration gradientActivity of P. luminescens chb compared with P. luminescens chb mutant00.511.522.533.5chb 100 ug/ml chb mutant 100 ug/mlAbsorbance 450nm1. Intro:The overall objective of this project is to generate novel carbohydrate bindingproteins for use in glycoprotein analysis which are amenable to large scaleproduction. The approach used here is the modification of prokaryoticglycolytic enzymes. The enzymes being studied are from Photorhabdusluminescens and are chitobiases (chb) which are a subset of glycolyticenzymes. These enzymes recognize and cleave N-Acetylglucosamine(GlcNAc) dimers to GlcNAc monomers.We will eliminate their enzymatic activity while hoping they still retain theirbinding capabilities. The resulting novel carbohydrate binding proteins willhave huge commercial potential in the field of glycoanalysis.Figure 1: A chitobiase from Serratiamarcescens. A chb from Serratiamarcescens has already been clonedand purified. Its catalytic activity hasbeen removed and it has beenshown to bind glycans in solution[Tews et al 1996, Prag et al 2000].Our specific objective is to look at ahomologue of this chb fromPhotorhabdus luminescens and usethe work on Serratia marcescens asa guide.2. Cloning, Expression and Purification of a chb from P.luminescensFigure 2: a) Construct map. Chb PCR product (yellow) was inserted into apQE60 vector as an NcoI-BamHI fragment. This vector adds a C terminal Histag (red) to its product. The vector also contains ampicillin resistance (blue) forselection. This product was subsequently transformed into Escherichia coliXL10Gold for expression. b) Samples from E. coli cultures containing theexpression vectors were taken every hour for 5 hours and lysed by sonication.SDS-PAGE was used to examine expression of chb c) IMAC purification of chba) b) c)4. Generation of a novel non-hydrolytic chb carbohydrate-binding protein.Activity of chb compared with it's commercial counterpart00.10.20.30.40.50.60.70.80.9Commercial Enzyme 10 ng/ml chb 10 ng/mlAbsorbance 450nm3. Enzymatic activity of thechb chitobiase comparedwith commercial counterpart00.20.40.60.811.21.4100ug/ml 1ug/ml 100ng/ml 10ng/ml GSL II ECLAbsorbance at 450nmLectinBinding of P. luminescens chb mutant to GlcNAc residues5. ELLA analysis detects binding of chb mutant to GlcNAcresiduesb)a)Figure 5: a) ELLA used to detect binding to GLcNAc residues using GSLIIas a positive control and ECL as a negative control. No binding by chbmutant to GlcNAc residues was detected however this could be due to aninability to detect a signal rather than no binding occurring. b) Competitiveassay using a constant concentration of GSLII with an increasingconcentration of chb mutant. Indirectly shows that chb mutant is binding toGlcNAc residues.Poster presentations•School of Biotechnology Research Day, DCU, January 2011•ISSC Review Meeting, DCU, June 2010• 3rd Annual Meeting of Glycoscience Ireland, UCD, August 2010.100kDaT0 T1 T2 T3 T4 T5 TF L FT W E1 E3 E5 E7 E9 E11Figure 4: Mutagenesis ofrecombinant chb results inabolition of catalytic activity.Figure 3: Colorimetric Assay totest activity of chb enzyme. Thesubstrate 4-Nitrophenol N-acetyl βD glucosaminide is cleaved by theenzyme to release a colouredcompound which can be detected.Oral Presentations• School of Biotechnology Seminar Series, DCU, April 2011

Glycolytic enzymes - novel carbohydrate binding proteins for glycoprotein analysis

DCU Online Research Access Service

•The cloning, expression, purification and characterisation of recombinant prokaryotic glycolytic enzymes
•The mutagenesis of prokaryotic glycolytic enzymes to generate novel recombinant carbohydrate binding proteins
•The characterisation of the binding profile of the novel recombinant carbohydrate binding protein

O\u27Connor, Brendan

Irish Universities

http://doras.dcu.ie/17765/1/Amy_ISSC_SFI_visit_June_2011_revised3.pdf

Glycolytic enzymes - novel carbohydrate binding proteins for glycoprotein analysis

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Irish Universities