The cloning , expression and characterisation of bacterial chitin-binding proteins from pseudomonas aeruginosa , serratia marcescens, photorhabdus luminescens and photorhabdus asymbiotica.

It is well recognised that most proteins are subject to post translational modifications and that these modifications can have specific effects on the biological properties and functions of these proteins. The majority of proteins secreted by cells are modified by the attachment of oligosaccharide chains. This glycosylation event has been shown to impact correct protein folding, protein stability, solubility, to aid in cell recognition and to help regulate cell processes. In order to gain a deeper understanding into the impact of altered glycosylation patterns on cellular processes and cell recognition it is necessary to develop new technologies to profile the glycan species displayed on the surface of protein molecules. The present study was dedicated to the development of prokaryotic chitin-binding proteins as novel carbohydrate-binding molecules. Prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus asymbiotica and Photorhabdus luminescens were cloned, over-expressed in E. coli and purified to homogeneity via (His)6 affinity tags. The activity and specificity of these proteins was tested using a number of insoluble substrates; chitin, chitosan and crystalline cellulose. The ability of these proteins to bind to protein linked glycans was tested using Enzyme linked lectin assays (ELLAs). None of the proteins exhibited any ability to bind glycoproteins in this assay format. A novel N-acetylglucosamine binding assay was developed using CBP21 and the ability to immobilise active CBP21 on a sepharose surface was also demonstrated. Sugar inhibition studies indicated that CBP21 may be capable of binding to mannan and galactan polymers. A site-directed mutagenesis of CBP21 was carried out on the putative binding domain residues to alter the affinity of CBP21. Residues Y54, E55, P56, Q57, S58, E60, T111, H114 and D182 were mutated to alanine, expressed, purified and characterised. The mutation H114A was shown to negatively impact on β-chitin affinity, the Q57A mutant had an increased affinity for chitosan with the proteins Y54A, T111A and D182A displaying an increased affinity for Cellulose. Furthermore it was shown that the putative C-terminal binding domain of CbpD is a chitin-binding domain and that the putative chitin-binding proteins CbpA and CbpL are capable of binding to both α- and β-chitin

The use of chitin binding proteins for glycoprotein analysis

The focus of the pharmaceutical industry has dramatically shifted in the past number of years. Traditional drugs were synthesised using chemical reactions have been replaced by recombinant glycoprotein molecules. These potential recombinant glycoprotein therapeutics display oligosaccharide structures on their surfaces that are recognised by their target host. The specific glycan moieties on the surface of the molecules vary dramatically and have a large impact on the efficacy of the drug. The development of bioanalytical tools to identify and separate the species of glyco-forms present in a preparation of the glycoprotein therapeutic will significantly help to advance the quality and effectiveness of recombinant glycoprotein molecules. Traditionally lectins, isolated from plants, had been used to profile sugar species displayed on glycoproteins. I have explored the use of prokaryotic chitin binding proteins (CBPs) to investigate structures on glycoproteins

Development of new bioaffinity phases for glycoprotein separation and analysis

The past decade has seen an appreciation of the critical biological significance of glycosylation and the impact that glycans can have on the efficacy, stability and immunogenicity of important glycoproteins. For the biopharma industry the level of product characterization and process monitoring demanded, particularly in the context of glycosylation, is set to increase and will become an even more significant issue with the entry of biosimilar products into the biopharmaceutical arena. In order to meet these demands of the regulatory biopharmaceutical and generic producers alike require rapid , sensitive and high throughput techniques to enable detailed glycoprotein separation and analysis. It is extremely difficult to separate closely related glycoforms using standard chromatographic methods, such as ion exchange chromatography (IEX), gel exclusion chromatography (GEC) and hydrophobic interaction chromatography (HIC). Here , we report the development of a series of new bio-affinity based phases that are capable of separating closely related glycoproteins/glycoproteins. These phases get their selectivity from a number of recombinant prokaryotic bioligands called lectins. Lectins are proteins that are capable of recognizing and binding reversibly to specific glycan structures. While lectin binding affinities for monosaccharides are generally low they bind to disaccharides and more complex oligosaccharide structures with significantly higher affinities and exquisite specificity

Exploiting prokaryotic chitin-binding proteins for glycan recognition

• The cloning, expression and characterisation of prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus luminescens Microfluidics and Photorhabdus asymbiotica • Development of an assay to assess the activity of chitin-binding proteins • Mutagenesis of chitin-binding proteins to alter glycan recognition pattern

Design of new highly functional polymer grafted polyhipes for proteins immobilization

PolyHIPE have proven to be useful in a large variety of applications included column filtration/separation, supported organic chemistry, as media for tissue engineering and 3D cell culture.1 The ability to conveniently modify pHIPE surfaces with functional groups is essential to opening new applications areas. The most promising method to conveniently modify pHIPE surface with a high density of functional groups is the “grafting from” approach. Stable polymer brushes covalently attached to the surface posses excellent mechanical and chemical robustness and offer the flexibility to introduce a large variety of functional monomers.2 We developed a new and unique pHIPE platform by incorporation of a polymerizable monomer with amino group into the HIPE available for different post in situ polymerization. The pHIPE with amino groups on the surface (pHIPE-NH2) can be directly used for the ring opening polymerization of amino acids N-carboxyanhydrates (NCAs) monomers to make pHIPE-g-polypeptide (such as pHIPE-g-poly(L-Benzyl Glutamate)) or easily converted to an atom transfer radical polymerization (ATRP) initiator for activators generated electron transfer (AGET) ATRP of tert-Butyl acrylate monomers. The polymers grafted can be deprotected to form pHIPE-g-poly(glutamic acid) or pHIPE-g-poly(acrylic acid) with reactive groups, on the surface of the pHIPE, available for further bioconjugation

Genetically enhanced recombinant lectins for glyco-selective analysis and purification

- Generation of a library of recombinant prokaryotic lectins (RPL’s) through random mutagenesis of the carbohydrate binding sites of bacterial lectins. - Characterisation of mutant lectins with respect to structure and specificity - Provision of mutant RPL’s with enhanced affinity and/or altered specificity, alongside wild-type RPL’s, for glycoprotein analysis and purificatio

Protein immobilization on highly functional polymer grafted polyHIPEs

PolyHIPE have proven to be useful in a large variety of applications included column filtration/separation, supported organic chemistry, as media for tissue engineering and 3D cell culture.[1] We developed a new pHIPE platform by incorporation of a polymerizable monomer with amino group into the HIPE available for different post polymerizations.[2] The pHIPE with amino groups on the surface (pHIPE-NH2) can be directly used for the ring opening polymerization of amino acids N-carboxyanhydrates (NCAs) to obtain pHIPE-g- polypeptides or easily converted to an atom transfer radical polymerization (ATRP) initiator for activators generated electron transfer (AGET) ATRP of tert-butyl acrylate. The polymers grafted were deprotected to form or pHIPE-g-poly(acrylic acid) with reactive groups on the surface of the pHIPE, available for bioconjugation of fluorescent proteins such as enhanced green fluorescent protein (eGFP

Polypeptide-grafted macroporous polyHIPE by surface-initiated N-Carboxyanhydride (NCA) polymerization as a platform for bioconjugation

A new class of functional macroporous monoliths from polymerized high internal phase emulsion (polyHIPE) with tunable surface functional groups was developed by direct polypeptide surface grafting. In the first step, amino-functional polyHIPEs were obtained by the addition of 4-vinylbenzyl or 4-vinylbenzylphthalimide to the styrenic emulsion and thermal radical polymerization. The obtained monoliths present the expected open-cell morphology and a high surface area. The incorporated amino group was successfully utilized to initiate the ring-opening polymer- ization of benzyl-L-glutamate N-carboxyanhydride (BLG NCA) and benzyloxycarbonyl-L-lysine (Lys(Z)) NCA, which resulted in a dense homogeneous coating of polypeptides throughout the internal polyHIPE surfaces as confirmed by SEM and FTIR analysis. The amount of polypeptide grafted to the polyHIPE surfaces could be modulated by varying the initial ratio of amino acid NCA to amino-functional polyHIPE. Subsequent removal of the polypeptide protecting groups yielded highly functional polyHIPE-g-poly(glutamic acid) and polyHIPE-g- poly(lysine). Both types of polypeptide-grafted monoliths responded to pH by changes in their hydrohilicity. The possibility to use the high density of function (−COOH or −NH2) for secondary reaction was demonstrated by the successful bioconjugation of enhanced green fluorescent protein (eGFP) and fluorescein isocyanate (FITC) on the polymer 3D-scaffold surface. The amount of eGFP and FITC conjugated to the polypeptide-grafted polyHIPE was significantly higher than to the amino- functional polyHIPE, signifying the advantage of polypeptide grafting to achieve highly functional polyHIPEs

Polypeptide-Grafted Macroporous PolyHIPE by Surface-Initiated <i>N</i>-Carboxyanhydride (NCA) Polymerization as a Platform for Bioconjugation

A new class of functional macroporous monoliths from polymerized high internal phase emulsion (polyHIPE) with tunable surface functional groups was developed by direct polypeptide surface grafting. In the first step, amino-functional polyHIPEs were obtained by the addition of 4-vinylbenzyl or 4-vinylbenzylphthalimide to the styrenic emulsion and thermal radical polymerization. The obtained monoliths present the expected open-cell morphology and a high surface area. The incorporated amino group was successfully utilized to initiate the ring-opening polymerization of benzyl-l-glutamate <i>N</i>-carboxyanhydride (BLG NCA) and benzyloxycarbonyl-l-lysine (Lys­(Z)) NCA, which resulted in a dense homogeneous coating of polypeptides throughout the internal polyHIPE surfaces as confirmed by SEM and FTIR analysis. The amount of polypeptide grafted to the polyHIPE surfaces could be modulated by varying the initial ratio of amino acid NCA to amino-functional polyHIPE. Subsequent removal of the polypeptide protecting groups yielded highly functional polyHIPE-<i>g</i>-poly­(glutamic acid) and polyHIPE-<i>g</i>-poly­(lysine). Both types of polypeptide-grafted monoliths responded to pH by changes in their hydrohilicity. The possibility to use the high density of function (−COOH or −NH₂) for secondary reaction was demonstrated by the successful bioconjugation of enhanced green fluorescent protein (eGFP) and fluorescein isocyanate (FITC) on the polymer 3D-scaffold surface. The amount of eGFP and FITC conjugated to the polypeptide-grafted polyHIPE was significantly higher than to the amino-functional polyHIPE, signifying the advantage of polypeptide grafting to achieve highly functional polyHIPEs