Day 3 :
- Track 5: Pharmacogenomics & Pharmacoproteomics
Track 6: Protein Purification
Chair
Cody Coyne
Mississippi State University, USA
Session Introduction
Cody Coyne
Mississippi State University, USA
Title: Necessary concepts in the design of the molecular structure and organic chemistry reactions for the synthesis of covalent biopharmaceuticals
Time : 10:00-10:25
Biography:
C P Coyne has completed his DVM at Colorado State University at Fort Collins Colorado, followed by an Internship in Surgery, PhD (Radiopharmacology) and Residency in Internal Medicine at the University of California at Davis. Currently he is a Professor of Molecular Pharmacology and Immunology in the Department of Basic Sciences, College of Veterinary Medicine at Mississippi State University. He has published over 50 peer-reviewed manuscripts in addition to the first comprehensive text on Diagnostic Pharmacology. Majority of recent and current on-going research investigations are devoted to the design of the molecular structure and organic chemistry reactions for the synthesis of biopharmaceuticals that have properties of selective targeted delivery and anti-neoplastic cytotoxicity.
Abstract:
Covalent bio-chemotherapeutics designed to possess properties of “targeted†delivery have significant merit due to their capability to selectively promote progressive intracellular chemotherapeutic accumulation and impose minimal chemotherapeutic exposure to healthy tissues and organ systems. Chemotherapeutics most extensively employed in organic chemistry reactions schemes for synthesizing covalent immunochemotherapeutics or other bio-chemotherapeutics with similar characteristics have been the anthracyclines and to a lesser extent, bleomycin, calicheamicin, chlorambucil, dexamethasone, fludarabine, gemcitabine, methotrexate, maytansinoids, monomethyl auristatin E, paclitaxel and vinca alkaloids. A limited spectrum of organic chemistry reaction schemes have been utilized to covalently bond chemotherapeutics and other pharmaceuticals to synthetic macromolecules, immunoglobulin, receptor ligands, glycoproteins, polysaccharides and lectins that can facilitate selective “targeted†delivery. Although methods have been described for synthesizing and purifying covalent bio-chemotherapeutics over prolonged reaction times, alternative methods and techniques have been developed that are efficient, rapid-in-duration, generate homogenous end-products with minimal side reactions and have modest requirements for advanced instrumentation. Critically important in the synthesis process of establishing the design of the molecular structure and organic chemistry reactions for synthesizing covalent immunochemotherapeutics is the selection of uniquely over-expressed sites on the external membrane of “targeted†cell populations. Ideally these sites should each facilitate selective “targeted†delivery, continual membrane deposition and progressive cytosol accumulation of a corresponding pharmaceutical moiety. Immunoglobulin as a carrier platform also affords the attribute of potentially promoting anti-neoplastic activity through both an anti-trophic effect and induction of multiple in vivo immune responses (e.g., antibody dependent cell cytotoxicity; complement cytolysis; opsonization).
Ryan Walsh
INRS–Institut Armand-Frappier Research Centre, Canada
Title: New views on old kinetic ideas: How empirical enzyme kinetic analysis can shed new light on disease mechanisms
Time : 10:25-10:50
Biography:
Ryan Walsh has completed his PhD in Chemistry from Carleton University (2012) and Masters in Anatomy and Neurobiology from Dalhousie University (2006). He is currently pursuing his Postdoctoral studies at the INRS-Institut Armand-Frappier Research Centre. His research interests range from enzyme kinetics to DNA nanotechnology and he has published more than 20 papers. His interest in enzyme kinetics has led to the publication of a book chapter on his enzyme kinetic theories and the proposal of a novel kinetic model of beta-amyloid processing by gamma-secretase in Alzheimer’s disease.
Abstract:
Enzyme kinetic analysis of drug interactions has remained the same for nearly a century and has essentially been supplanted by the use IC50 and EC50 in drug analysis. The fall from favor of enzyme kinetic analysis can be primarily attributed to the difficulties associated with kinetic modeling and the absence of relevance, inhibitory classifications, have on therapeutic development. However, the problems with enzyme kinetic analysis can also be attributed to a lack of clear distinction between binding constants and terms defining the effect produced by the compounds under investigation. The most basic inhibitory equations; competitive, non-competitive and mixed non-competitive inhibition, define inhibitory effect using the disassociation constant (Ki) and this necessitates the use of separate equations to model the inhibitory effects attributed to each equation. By designating the Ki as simply, a binding term like the Kd in receptor interactions the effects compounds have on enzyme activity can be defined separately producing a simple empirical equation for activators and inhibitors. This treatment unifies and simplifies kinetic analysis providing an intuitive way of conceptualizing the modulation of complex catalytic regulatory processes. For example, the modulation of substrate activation and substrate inhibition associated with amyloid precursor protein processing by gamma-secretase can be simple, concise manner. While IC50 values may be sufficient for characterizing the majority of drug interactions, the complex interactions at the center of diseases that have stubbornly resisted therapeutic progress may benefit from this empirical modeling approach.
Rimantas Slibinskas
Vilnius University, Lithuania
Title: Generation of purely native recombinant human cellular proteins and human viral antigens in the yeast expression systems
Time : 11:05-11:30
Biography:
Rimantas Slibinskas has completed his PhD in 2006 from Vilnius University in Lithuania. He is Senior Research Associate at the Institute of Biotechnology (IBT) of Vilnius University and the Director of biotechnology company UAB Baltymas, a spin-off of the IBT, specialising in production of recombinant proteins for diagnostic and therapeutic use. He has published more than 15 papers in reputed journals and is co-author of international patent applications/inventions in the field of recombinant protein expression in yeast cells. Currently, he leads research projects granted by EU in the field of proteomics and genetic engineering of yeasts.
Abstract:
Despite many advantages, the potential of yeast expression systems has not been entirely exploited and most recombinant proteins are currently produced in mammalian cells or prokaryotes. Here we show a substantial progress in production of native recombinant human cell proteins in yeasts. During past decade we produced a range of human virus nucleocapsid proteins assembled in nucleocapsid like particles (NLP) including measles, mumps, respiratory syncytial virus, different types of parainfluenza and influenza virus NLPs. It was shown that yeast-expressed NLPs are better diagnostic reagents for diagnosis of viral infections than the same proteins produced in bacteria. Therefore, these recombinant protein products are already being introduced in antigen diagnostic market. Expression of secretory human proteins in yeasts is usually less successful than for those synthesized in cytoplasm like viral nucleoproteins. Evolution of cells from lower eukaryotes to higher multicellular organisms including mammalians was mostly focused on the evolving mechanisms in the secretory pathway. Recently we found that human endoplasmic reticulum (ER) chaperones GRP78/BiP, ERp57 and calreticulin are correctly processed and efficiently secreted from yeast cells using their own native signal sequences. Compatibility of signal peptidase complexes in yeast and human cells, and inability of yeasts to retain human ER chaperones inside the cell makes such expression system unique for high-level production of active human BiP, ERp57 and calreticulin. In conclusion, here we demonstrate that yeast expression systems can generate purely native recombinant human cell proteins, which are purified to high purity without adding any tags or changing native amino acid sequence.
Ikram ul Haq
GC University, Pakistan
Title: Cloning, characterization and saccharification analysis of GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host
Time : 11:30-11:55
Biography:
Ikram ul Haq (SI) has completed his PhD at the age of 40 years from The University of Punjab Pakistan and postdoctoral studies from Cornell University, USA. He is serving as director research, innovation and commercialization GC University, Lahore. He was the Dean Science and technology, GC University, Lahore. He also served as Director Institute of Industrial biotechnology, GCU Lahore and currently working as Professor Emeritus in the same institute. He is a fellow of Pakistan Academy of Science, Fullbrighter, President FABA Lahore Chapter, President Pakistan botanical society. He has published more than 300 papers in reputed journals and has been serving as an editorial board member so many reputed journals.
Abstract:
The increasing demand of energy has strongly stimulated the research on conversion of lignocellulosic plant biomass by the action of cellulases enzymes into reducing sugars, for the subsequent production of bioethanol. Endoglucanases are mainly responsible for hydrolyzing the internal glycosidic bond to decrease the length of the cellulose chains. Obtaining efficient and thermostable endoglucanase has become the goal of much research worldwide. Therefore, our research work was focus to search for new resources of endoglucanases, which was thermostable and with high catalytic efficiency. The article focuses on the thermotolerant endo-1,4-β-glucanasegene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus for its potential usage for the hydrolysis of lignocellulosic biomass and in different industrial applications. Thermostable endoglucanase can be used simultaneously and directly in the saccharification procedure without a pre-cooling process of biomass. Purified enzyme was optimally active with 530 Umg-1 of specific activity against CMC at pH 6.0 and 95°C, which has exhibited a half- life (t1/2) of 6.6 min even at temperature as high as 97°C and stable upto 8h at 80°C. The recombinant enzyme saccharified pre-treated wheat straw and baggase to 3.32% and 3.2%, respectively after 6h incubation at 85°C. Its thermostability, resistance to heavy metal ions and high specific activity make endoglucanase a potential and promising candidate for various industrial applications such as in textile industry (in biostoning and biofinishing), in animal feed production, in processing of beer and fruit juice, in biomass hydrolysis (bioethanol production) and paper industry.
Irfana Mariam
Queen Mary College Lahore, Pakistan
Title: Purification and characterization of glucose oxidase from locally isolated strain of Aspergillus niger
Time : 11:55-12:20
Biography:
Irfana Mariam has completed her PhD at the age of 35 years from The University of Punjab Pakistan and postdoctoral studies from Cornell University, USA. She is serving as Principal Queen Mary College Lahore Punjab Pakistan. She has published 35 research publications. She served as Assistant Professor in the GC University Lahore Punjab Pakistan.
Abstract:
Aspergillus niger strain IIB-247 was used for production of Glucose oxidase using medium containing glucose 6%, peptone 0.3%, (NH4)2HPO4 0.04%, KH2PO4 0.0188%, MgSO4.7H2O 0.0156%, CaCO3 3.5%. Maximum production (12.12 ± 0.01 U/mg) was obtained at pH 7 and temperature and 30°C respectively after 72 hours of fermentation. Optimum glucose oxidase production coincided with glucose depletion (87.5%). Ammonium sulfate precipitation and Ion exchange chromatography resulted in 53.5 % yield and 16. 81 fold purification with enhancement of specific activity (203.56 ± 0.02 U/mg). Kinetic characterization of enzyme revealed D-Glucose as highly specific substrate for enzyme with Km value of 30.5 mM. Thermodynamic evaluation of enzyme revealed activation energy (Ea) as 13.14 KJ/mol, enthalpy of activation (ΔH) as 10.87KJ/mol and entropy of activation (ΔS) as -1.139 KJ/mol respectively. The enzymes catalytic activity was observed to be reduced by some heavy metals such as i.e. Hg2+, Pb2+, Cu+ and Cd2+. The enzyme remained stable at pH 6. Maximum shelf life of the enzyme was observed in lyophilized form at -20°C for a period of 2 months. All of the above mentioned characters of GOX made it a potential candidate in food industry for residual glucose and oxygen removal in foods and beverages in order to prolong their shelf lifes. Glucose oxidase can also be used to remove oxygen from the top of bottled beverages before they are sealed. In addition, glucose oxidase is used to prevent colour and flavour loss from foods and beverages. Apart from that it can be used for estimation of blood glucose level as well.
Anuradha S. Nerurkar
The Maharaja Sayajirao University of Baroda, India
Title: Biotechnological applications of Functional Bacterial Amyloid (FuBa) proteins of gram positive bacteria
Biography:
Anuradha S Nerurkar has completed her MSc (Microbiology) PhD from RTM Nagpur University, Maharashtra, India. She is teaching in the Department of Microbiology & Biotechnology Centre of The M.S. University of Baroda since 1996 and she is currently a Professor of Microbiology. Her areas of research are bacterial amyloids and biotechnological applications, bacterial bio-emulsifier and its eco-physiological role and Quorum quenching approach of bio-control of plant pathogens. She has published 22 papers in reputed journals and written five book chapters.
Abstract:
Functional bacterial amyloids are a group of proteinaceous surface structures that are resistant to many enzymes, chemicals and thermal denaturants. Amyloid formation is hallmark of certain human diseases like Alzheimer’s, Huntington’s and Prion diseases. However, in bacteria the amyloids have been found to be biologically functional molecules. Significance of these structures that include fimbriae and other appendages in Gram negative bacteria like E. coli and Psuedomonads have been studied in depth. Biotechnogical applications of amyloid proteins have not been exploited as yet. In our two separate studies Gram positive bacterial amyloid proteins have been found to be associated with interesting biotechnological characteristics. Solibacillus silvestris AM1, an estuarine isolate has been found to produce a bio-emulsifier that emulsifies diverse hydrocarbons. The biochemical characterization of this bio-emulsifier revealed it to be an amyloid protein of 30 kDa subunit which aggregates into fibrils as observed in electron micrographs. Purification and further characterization of this confirmed it to be related to flagellar proteins. In another study amyloid protein producing Sporosarcina sp. CR4 isolated from aeration tank of sewage treatment plant was found to possess flocculating activity. Purification and characterization of this bioflocculant was also carried out. Flocculation activity has been demonstrated to present in the amyloid protein.