The overall objective of this project is to generate novel carbohydrate binding proteins for use in glycoprotein analysis which are amenable to large scale production. The approach used here is the modification of prokaryotic glycolytic enzymes. Their enzymatic activity will be eliminated while hoping they still retain their binding capabilities. These proteins will be immobilized onto different surfaces to generate advanced bioanalytical platforms which will have huge commercial potential in the field of glycoanalysis

Clarke, Paul A.

Harrington, Amy

O'Connor, Brendan

Thompson, Roisin

English

O\u27Connor, Brendan

Irish Universities

The study of a prokaryotic glycolytic enzyme

DCU Online Research Access Service

00.10.20.30.40.50.60.70.8Glycoprotein Glycoprotein w ith sugar of interestremovedAbsorbance 450nmThe Study of a Prokaryotic Glycolytic Enzyme                                           Amy Harrington1, Roisin Thompson1, Brendan O’Connor1, Paul Clarke 1                                                                                                   1. Irish Separation Science Cluster, Dublin City University, Dublin                                                                           This research has been funded by Science Foundation Ireland under grant number 08/SRC/B1412  Objectives: • The cloning, expression, purification and characterisation of a recombinant prokaryotic glycolytic enzyme  • The site specific mutagenesis of the prokaryotic glycolytic enzyme to generate a novel recombinant carbohydrate binding protein  • The characterisation of the binding profile of the novel recombinant carbohydrate binding protein • Random Mutagenesis of the glycolytic enzyme to study its mechanism of action and the possible generation of more novel carbohydrate binding proteins • Immobilisation of the protein to various surfaces including monoliths, polyHIPES and gold nanoparticles to produce advanced bioanalytical platforms  5. Future Work 1. Introduction:  The overall objective of this project is to generate novel carbohydrate binding proteins for use in glycoprotein analysis which are amenable to large scale production. The approach used here is the modification of prokaryotic glycolytic enzymes. Their enzymatic activity will be eliminated while hoping they still retain their binding capabilities. These proteins will be immobilized onto different surfaces to generate advanced bioanalytical platforms which will have huge commercial potential in the field of glycoanalysis.  Figure 1: A homologue of the prokaryotic glycolytic enzyme. A homologue of the prokaryotic glycolytic enzyme has already been cloned and purified. Its catalytic activity has been removed and it has been shown to bind glycans in solution [Tews et al. 1996, Prag et al. 2000]. This work has been used as a template for our site specific mutagenesis.  2. Cloning, Expression, Purification and Characterization of the prokaryotic glycolytic enzyme (G) Figure 2: a) Construct map. PCR product of the gene of interest (yellow) was inserted into a pQE60 vector. This vector adds a C terminal His tag (red) to its product. The vector also contains ampicillin resistance (blue) for selection. This product was subsequently transformed into Escherichia coli KRX for expression.  a) b) c) 3. Site Specific Mutagenesis of the glycolytic enzyme (G) to remove activity and generate a novel carbohydrate binding protein (∆G) Figure 3: Site Specific Mutagenesis of recombinant enzyme G results in a significant reduction of catalytic activity as determined by the colorimetric assay. 4. ELLA analysis detects binding of carbohydrate binding protein (∆G) to sugar residues b) a) Figure 4: a) ELLA used to detect binding of ∆G to sugar of interest.  A sandwich assay format was used as direct detection was not successful. b) Binding of ∆G to sugar of interest is concentration dependent • Random mutagenesis to: • Study mechanism of action of the enzyme • Generate more novel carbohydrate binding proteins • Immobilisation of carbohydrate binding protein (∆G) to various surfaces including monoliths, polyHIPES and gold nanoparticles to generate advanced bioanalytical platforms. 100kDa    T0  T1  T2   T3   T4   T5   TF     L   FT  W  E1 E3 E5 E7 E9 E11 Modification of the enzyme to generate novel carbohydrate binding proteins for glycoprotein analysis y = 0.0987x + 0.1276R2 = 0.986400.20.40.60.811.2-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 31/S1/Vd) b) Supernatant samples from E. coli cultures containing the expression vectors were taken every hour for 5 hours. SDS-PAGE was used to examine expression of the enzyme (G). c) IMAC purification of the enzyme (G). d) Colorimetric Assay to test activity of the glycolytic enzyme (G). A 4-Nitrophenol linked substrate was cleaved by the enzyme to release a coloured compound which can be detected. A Lineweaver-Burke plot was generated with the results. e) Lineweaver Burke values calculated to describe activity of the enzyme (G).  00.511.522.533.5G	  100	  ug/ml ∆G	  100	  ug/ml00.10.20.30.40.50.60 5 10 15 20 25 30 35Concentration of Glycoprotein (ug/mL)Absorbance 450nme) c) Figure 5: a) and b) Lectin modified gold nano-particles on a polymer monolithic phase. c) GFP immobilised onto polyHIPES.  Vmax (mM) 7.83 Km (mM) 0.75 Kcat (s-1) 0.783 Kcat/Km (1/mM.s) 1.044 

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The study of a prokaryotic glycolytic enzyme

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