Day 1 :
Keynote Forum
R Stephen Berry
The University of Chicago, USA
Keynote: The topographies of energy landscapes of real proteins
Time : 09:35-10:20
Biography:
R Stephen Berry has completed his undergraduate and graduate studies at Harvard. He was a Member of the Chemistry Faculty at The University of Michigan then at Yale and from 1964 at The University of Chicago. His research has covered a wide range within physical chemistry, from protein dynamics to atomic scattering and photo-ionization, to atomic and molecular clusters and to finite-time thermodynamics.
Abstract:
Exploration of the energy landscapes of two relatively small proteins, protein G and srcSH3 and of two random peptides containing the same amino acids reveals the kind of topography that can be expected for real proteins, the topographies that proteins must traverse as they fold to their native states. The most significant characteristic of those landscapes is probably the multiplicity of funnels that they display; the simple single “folding funnel†model is quite unlike the energy landscapes of these real proteins. The folding processes that bring these proteins to their native state follow varieties of trajectories, some of which avoid the “wrong†funnels while others visit those misfolded structures and then leave them and go to the funnel of the native state. A network analysis reveals some of what seem to be common characteristics of the folding process.
Keynote Forum
David Mead
Lucigen Corporation, USA
Keynote: A novel platform for expressing multi-subunit protein complexes and pathways
Time : 10:20-11:05
Biography:
David Mead leads research and development efforts for the Company’s research use only products. He earned his PhD in Physiology and Biophysics at the University of Illinois–Champaign/Urbana. He is the Inventor of TA cloning and he is the co-author of fifty two publications.
Abstract:
Circular plasmids frequently are difficult to use for expression of multiple or large genes, repetitive or toxic proteins and pathways or multi-component gene circuits. The inherent supercoiling of circular plasmids imparts instability forrnthese and other complex gene sequences. The pJAZZ plasmid is a unique linear cloning vector that tolerates nearly any DNA sequence including AT-rich genes and highly repetitive sequences that are impossible to capture in typical circular vectors. pJAZZ has a cloning capacity of up to 40 kb and lacks the cloning bias inherent to circular plasmids, enabling the assembly of complex multi-gene systems. We are developing pJAZZ expression vectors for a variety of applications including expression of mega Dalton proteins, expression of multiple proteins (up to six currently) and production of metabolites from prokaryotic pathways. The vector includes improved light-inducible expression cassettes that have distinct advantages over small-moleculerninduction such as: Precise spatial control of expression; instantaneous initiation and termination of inducing agent; tunability and lack of toxicity or cross-reactivity. The light inducible gene circuit has proven to be significantly more efficient in the linear pJAZZ backbone than in circular vectors.
Keynote Forum
Ram Samudrala
State University of New York, USA
Keynote: Interactomics: Computational analysis of novel drug opportunities
Time : 11:25-12:10
Biography:
Ram Samudrala is Professor and Chief, Division of Bioinformatics, State University of New York, Buffalo researching multi scale modeling of atomic, molecular, cellular and physiological systems with an emphasis on protein and proteome structure, function, interaction, design and evolution. His work has led to more than 115 publications in journals such as Science, Nature, PLoS Biology, the Proceedings of the National Academy of Sciences and the Journal of the American Medical Association. He has joined the University of Washington Faculty in 2001 (where he remains as an Affiliate Professor) after completing his Doctoral research with John Moult at the Center for Advanced Research in Biotechnology in 1997 and his Postdoctoral research with Michael Levitt (2013 Nobel in Chemistry) at Stanford University in 2000, which resulted in him making some of the best predictions at the first three community-wide assessment of protein structure prediction (CASP) experiments.
Abstract:
We have developed a Computational Analysis of Novel Drug Opportunities (CANDO) platform funded by a 2010 NIH Director’s Pioneer Award that analyses compound-proteome interaction signatures to determine drug behaviour, in contrast to traditional single target approaches. The platform uses similarity of interaction signatures across all proteins as indicative of similar functional behaviour and non-similar signatures (or regions of signatures) as indicative of off- and anti-target (side) effects, in effect inferring homology of compound/drug behaviour at a proteomic level. We have created a matrix of predicted interactions between 3,733 human ingestible compounds (including FDA approved drugs and supplements) × 48,278 proteins using our hierarchical chem and bio-informatic fragment-based docking with dynamics protocol (from over one billion predicted interactions total). We applied our compound-proteome signature comparison and ranking approach to 2030 indications with one approved compound and yielded benchmarking accuracies of 12-25% for 1439 indications with more than approved compound. We are prospectively validating \\\"high value\\\" predictions in vitro, in vivo, and by clinical studies for more than forty indications, including dental caries, dengue, tuberculosis, ovarian cancer, cholangiocarinomas, among many fothers. 58/163 (36%) predictions over twelve studies across ten indications show comparable or better activity to existing therapies, or micromolar inhibition at the cellular level, and serve as novel repurposeable therapies. Our approach is applicable to any compound beyond those approved by the FDA, and also include can readily consider mutations in protein structures to enable personalization based on genotype, foreshadowing a new era of faster, safer, better and cheaper drug discovery.
- Track 1: Enzyme Engineering
Track 2: Recombinant Protein Expression
Chair
Frits Goedegebuur
DuPont Industrial Biosciences
The Netherlands
Co-Chair
Sarah Morais
Weizmann Institute of Sciences, Israel
Session Introduction
Kelley Moremen
University of Georgia
USA
Title: Strategies for recombinant production of human glycosylation enzymes: Comparison of mammalian and insect cell expression systems
Time : 12:10-12:35
Biography:
Kelley Moremen, PhD, is Distinguished Research Professor of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia. He received his PhD at Vanderbilt University and Postdoctoral training at MIT. Dr. Moremen is a leader in the structure, function, roles, and regulation of enzymes involved in mammalian glycan biosynthesis and catabolism. The presentation will discuss the development of a recombinant platform for production of all mammalian glycosyltransferases, glycosidases, and sulfotransferases (target gene list of >350 coding regions) in baculovirus, and mammalian cells to facilitate biochemical, enzymatic and structural studies on theenzymes and applications to chemoenzymatic synthesis.
Abstract:
Glycan structures on cell surface and secreted glycoproteins play critical roles in biological recognition and targeting events in animal systems. The enzymes that synthesize these structuresreside in membranes of the secretory pathway and modify glycoproteins in transit to the cell surface. These enzymes are generally poorly understood largely because they are challenging targets for functional expression even in eukaryotic hosts. We have established library of expression constructs encoding all human glycosylation enzymes (target gene list of ~350 coding regions) as secreted catalytic domain fusion proteins for recombinant production in HEK293 cells and baculovirus-infected insect cells. Comparison of expression levels in each host system indicates similar trends where most proteins are well-expressed but only a subset are efficiently secreted. However, many of the enzymes are more effectively produced and secreted in one recombinant host system or the other suggesting that differences in host secretory machinery can influence the yield of recombinant products. This presentation will summarize our strategies for expression and downstream workflows for biochemical and structural studies with goals to advance our understanding of the enzymatic machinery for glycan synthesis and modification.
Frits Goedegebuur
DuPont Industrial Biosciences, The Netherlands
Title: IPE - Industrial protein engineering: Accelerated evolution
Time : 12:35-13:00
Biography:
Frits Goedegebuur started in industrial biotechnology in 1988 at Gist Brocades and is now, via Genencor International (1995), Danisco (2005), working for DuPont Industrial Biosciences (2011) as Senior Scientist. He is a member of DuPont’s R&D management team and is project leader of a global protein engineering research project. He has filed ~120 patents, of which ~60 patents have been granted.
Abstract:
Historically, the protein design process has been approached as equivalent to the problem of finding in all of protein space the one best sequence for a single property. For accelerated evolution, the problem to be solved is to identify at least one protein sequence that meets or exceeds the minimum value required for a number of properties. Protein engineered proteins came about through accumulation of point mutations that were not deleterious for any important property. Using the “Super-screen†process, we can determine all those mutations in a matter of weeks for any protein for all important properties. Combining these mutations in the proper way can rapidly produce a protein with all desired properties.
Janusz Bujnicki
International Institute of Molecular and Cell Biology, Poland
Title: Engineered “restriction RNases†for sequence-specific cleavage of dsRNA and RNA in DNA-RNA hybrids
Time : 14:00-14:25
Biography:
Janusz M Bujnicki is a Professor of Biology and Head of a Research Group at IIMCB in Warsaw and at Adam Mickiewicz University, Poland. He has graduated from the Faculty of Biology, University of Warsaw in 1998 and defended his PhD in 2001. He was awarded with Habilitation in 2005 and with the Professor Title in 2009. His research combines bioinformatics, structural and synthetic biology. He is an author of >280 publications which have been cited >5300 times (self-citations excluded), Hirsch index 37 (according to Scopus). He has received numerous awards, prizes, fellowships and grants including EMBO/HHMI Young Investigator Program award, ERC Starting Grant, Award of the Ministry of Science and Award of the Prime Minister and was decorated with the Knight's Cross of the Order of Polonia Restituta by the President of the Republic of Poland. In 2013 he has won the national plebiscite “Poles with Verve†in the Science category. He is involved in various scientific organizations and bodies including the Polish Young Academy, Citizens of Science, Science Europe and Scientific Policy Committee. He is also an Executive Editor of the Journal Nucleic Acids Research.
Abstract:
Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Thus far, there have been no RNases available for purely sequence-dependent fragmentation of RNA, analogous to restriction enzymes for DNA. We have therefore searched for existing RNases that could be engineered to become sequence-specific. Using a combination of bioinformatics methods and experimental protein engineering we have obtained prototypes of two sequence-specific “restriction RNases†(RRNases): First, we identified members of the RNase III super family that exhibit sequence specificity towards dsRNA: They recognize a specific tetranucleotide target sequence and are capable of cleaving individual sites in long dsRNA molecules. For one of such enzymes we solved the crystal structure and constructed a structural model of a protein-RNA complex. Second, a prototype RRNase that cleaves the RNA strand in DNA-RNA hybrids five nucleotides from a nonanucleotide recognition sequence was constructed by fusing two functionally distinct domains: A non-specific RNase HI and a zinc finger that recognizes a sequence in DNA-RNA hybrids. The optimization of the fusion enzyme specificity was guided by a structural model of the protein-substrate complex and involved a number of steps including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Potentially, RRNases may be used in vitro for production of RNA molecules with defined length and termini which may be a cheaper alternative to chemical synthesis; they may be also used in vivo for targeted RNA degradation.
David Mead
Lucigen Corporation, USA
Title: Novel solubility fusion partners high throughput system to produce soluble proteins
Time : 14:25-14:50
Biography:
David Mead leads research and development efforts for the Company’s research use only products. He earned his PhD in Physiology and Biophysics at the University of Illinois–Champaign/Urbana. He is the Inventor of TA cloning and he is the co-author of forty four publications.
Abstract:
A large fraction of heterologous proteins are insoluble or poorly expressed in Escherichia coli. One solution to this problem is to fuse a “solubility tag†to the target protein. Selection of the best tag is a time consuming trial-and-error process that requires testing multiple different promoters, strains, and cloning technologies. Lucigen has developed a simple solution to simultaneously test multiple tags within the context of a single promoter, vector and host system. Lucigen’s Solubility Panel consists of multiple cleavable fusion partners within a robust enzyme-free cloning platform. In addition, a novel yellow fluorescent protein significantly enhances solubility and expression while providing an instant visual report of the amount of soluble, active protein. This system permits rapid, simultaneous screening of multiple factors demonstrated to improve solubility and/or expression in a high throughput format.
Ram Samudrala
State University of New York, USA
Title: Multiscale modelling of relationships between protein classes and drug behavior across all diseases using the cando platform
Time : 14:50-15:15
Biography:
Ram Samudrala is a Professor and Chief, Division of Bioinformatics, State University of New York, Buffalo researching multi scale modeling of atomic, molecular, cellular and physiological systems with an emphasis on protein and proteome structure, function, interaction, design and evolution. His work has led to more than 115 publications in journals such as Science, Nature, PLoS Biology, the Proceedings of the National Academy of Sciences and the Journal of the American Medical Association. He has joined the University of Washington Faculty in 2001 (where he remains as an Affiliate Professor) after completing his Doctoral research with John Moult at the Center for Advanced Research in Biotechnology in 1997 and his Postdoctoral research with Michael Levitt (2013 Nobel in Chemistry) at Stanford University in 2000, which resulted him in making some of the best predictions at the first three community-wide assessment of protein structure prediction (CASP) experiments.
Abstract:
We have examined the effect of eight different protein classes (channels, GPCRs, kinases, ligases, nuclear receptors, proteases, phosphatases and transporters) on the benchmarking performance of the CANDO drug discovery and repurposing platform. The first version of the CANDO platform utilizes a matrix of predicted interactions between 48,278 proteins and 3733 human use compounds that map to 2030 indications/diseases using a hierarchical chem and bio-informatic fragment based docking with dynamics protocol. The platform uses similarity of compound-proteome interaction signatures as indicative of similar functional behavior and benchmarking accuracy is calculated across 1439 indications/diseases with more than one approved drug. The CANDO platform yields a significant correlation (0.99, p-value <0.0001) between the numbers of proteins considered and benchmarking accuracy obtained indicating the importance of multi-targeting for drug discovery. Average benchmarking accuracies range from 6.2% to 7.6% for the eight classes when the top 10 ranked compounds are considered in contrast to the range from 5.5% to 11.7% obtained for the comparison/control sets consisting of 10, 100, 1000 and 10000 single best performing proteins. These results are two orders of magnitude better than the average accuracy of 0.2% obtained when using randomly generated matrices. Different indications perform well when different classes are used but the best accuracies (11.7%) are achieved with a combination of classes consisting of the broadest distribution of protein folds. Our results illustrate the utility of the CANDO approach and the consideration of different protein classes for devising indication specific protocols for drug repurposing as well as drug discovery.
Muhammad Waheed Akhtar
University of the Punjab, Pakistan
Title: Engineering glycosyl hydrolases using binding modules optimally
Time : 15:15-15:40
Biography:
Muhammad Waheed Akhtar is currently a Professor Emeritus in University of the Punjab, Pakistan. His current research interests include engineering cellulases and xylanases and their over-expression to construct a potent enzyme mixture for saccharification of pre-treated plant biomass. His group is also working on designing fusion antigens for a reliable sero diagnosis of tuberculosis. He has supervised research of several dozens of successful PhD graduates and published over 150 research papers.
Abstract:
Vast advances in recombinant DNA technology combined with the large variety of protein engineering methods, in silico tools and high-throughput screening techniques allow unlimited possibilities of improving proteins and enzymes for applications. Glycosyl hydrolases commonly contain the carbohydrate binding modules (CBMs) through which they bind to the substrates thus facilitating the catalytic domain (CD) to act. However, the role of the CBMs in the activities of cellulases and xylanases seems quite varied. For example, in the case of the endoglucanase CelA of Clostridium thermocellum the binding module CBM3a when attached to the n-terminal of the CD is more than twice as active as the variant having the binding module attached to the c-terminal of the CD. In the case of xylanase XynZ of C. thermocellum deletion of CBM6 which is found in association with the CD in the native state enhances the activity manifold. However, the addition of CBM22 to the CD enhanced the activity. Molecular modeling analyses of the structures show that for optimal activity of the enzyme orientation of the catalytic and the binding residues of the CD and the CBM respectively should lie in a compatible orientation. Also active site residues of the CD when in association with the CBM must be readily available to the substrate. Thus the CBMs are not only specific for binding to the substrate but their orientation in association with the CD is also important for activity.
Mridul Mukherji
University of Missouri-Kansas City, USA
Title: A mechanistic overview of TET-mediated 5-methylcytosine oxidation
Time : 16:00-16:25
Biography:
Mridul Mukherji has completed his PhD from Oxford University, UK and Postdoctoral studies from the Scripps Research Institute, USA. He works at the University of Missouri at the rank of an Associate Professor. He has published more than 30 papers in reputed journals and serves as an Editorial Board Member of 4 international journals.
Abstract:
Methylation of DNA at the carbon-5 position of cytosine plays crucial roles in the epigenetic transcriptional silencing during metazoan development. Recent identification of Ten-Eleven Translocation (TET)-family demethylases have added a new dimension to dynamic regulation of 5-methylcytosine (5mC), and thus, inheritable and somatic gene silencing. The interest in hematology was particularly stimulated by the recent discovery of TET2 mutations in myeloid malignancies which were proven to be leukemogenic in murine knockout models. The TET-family enzymes are Fe(II), 2-oxoglutarate-dependent oxygenases and catalyze demethylation of 5mC by iterative oxidation reactions. In the last decade results from numerous studies have established a key role for these enzymes in epigenetic transcriptional regulation in eukaryotes primarily by hydroxylation reactions. The TET catalyzed hydroxylation and dehydration reactions in the mammalian system exemplify the diversity of oxidation reactions catalyzed by Fe(II), 2-oxoglutarate-dependent oxygenases, and suggest an existence of other types of oxidation reactions catalyzed by these enzymes in the eukaryotes, which are so far only documented in prokaryotes. Here, we will describe the TET-mediated 5mC oxidation in light of the putative reaction mechanism of Fe(II), 2-oxoglutarate-dependent oxygenases.
Barindra Sana
A*STAR, Singapore
Title: Development of whole cell biosensor for screening of engineered lignin-degrading enzymes
Time : 16:25-16:50
Biography:
Barindra Sana is a Research Scientist at the Agency for Science Technology And Research (A*STAR), Singapore. He has completed his PhD from Jadavpur University, India and persued his Postdoctoral Research at Nanyang Technological University, Singapore. His current research area includes protein/enzyme engineering, biomass conversion and industrial biotechnology. He has also research interest in several inter-disciplinary fields of biosciences and bioengineering including nanobiotechnology, environmental biotechnology and biomedical engineering. He has published more than 20 peer-reviewed articles, book chapters and he has also been serving as Editorial Board Member of two scientific journals.
Abstract:
Lignin is a potential renewable raw material for synthesis of various value-added chemicals that can substitute fossil-derived consumer products. Huge lignin biomass is produced as by-product of paper industry while cellulosic components of plant biomass are utilized in paper pulp. Instead of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex cross-linked three dimensional structures that cannot be efficiently degraded by currently available enzymes. Effective lignin degrading enzymes could be developed by enzyme engineering. Directed evolution is a strong tool for enhancing activity of currently available enzymes but application of this technique for improving efficiency of lignin degrading enzyme is limited due to unavailability of any high throughput screening method. With an objective of detecting the lignin degradation products, we identified two E. coli promoters that are upregulated by potential lignin degradation products (e.g., vanillin, acetovanillone, guaiacol and veratraldehyde) and recombinantly placed a ‘very green fluorescence protein’ (vGFP) gene under control of these promoters within a customized plasmid. The whole cell sensor was developed by transforming E. coli cell with these constructs. Although the constructs showed some leaky expression, the cells responded by increasing fluorescence while grown in the presence of the lignin degradation products which was detected by FACS. The response was dose-dependent. Both sensors showed best response to vanillin as an individual compound but better response was achieved with the mixture of the lignin degradation products at a certain composition. Once the problem of leaky expression is solved, this technique could be potentially used for directly screening of libraries grown on agar plate.
Sarah Morais
Weizmann Institute of Sciences, Israel
Title: Improved bacterial enzyme systems for deconstruction of cellulosic biomass for production of liquid biofuels
Time : 16:25-16:50
Biography:
Sarah Morais, a biotechnological engineer from France, has completed her PhD at the age of 28 years from the Hebrew University of Jerusalem (Israel) and Postdoctoral studies from the Weizmann Institute of Sciences, Israel. She is now employed as an Academic Senior Intern at the Weizmann Institute of Sciences. She has published 13 papers in reputed journals and wrote 4 peer-reviewed book chapters.
Abstract:
The major objective of my research is to employ the designer cellulosome concept for improved enzymatic conversion of recalcitrant plant-derived cellulosic biomass into soluble sugars as an interim feedstock for production of liquid biofuels. The enzymes required for conversion of biomass into fuel on the industrial scale represent a major bottleneck for cost-effective replacement of fossil-based fuels. Thermostable cellulases and associated enzymes that degrade plant cell wall polysaccharides offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. To reduce enzyme costs, we improved the thermostability and durability characteristics of representative enzymes, thus increasing their overall activity during the deconstruction of lignocellulosic biomass. For this purpose we employed directed evolution and knowledge-based library design of selected enzymes. The improved enzymes will be incorporated into designer cellulosome complexes and their performance on recalcitrant lignocellulosic substrates will be evaluated with reference to complexes containing the original unmodified enzymes and to commercial enzymatic cocktails. The enzymes to be used include endoglucanases, exoglucanases and ï¢-glucosidase– all of which are key enzyme components of cellulosomes and together act synergistically in the deconstruction of the cellulosic part of the plant biomass. The improvements gained by this novel strategy are expected to yield highly active robust enzymes and consequent cost reduction in the conversion of biomass into soluble fermentable sugars en route to biofuel production.
- Track 3: Protein Engineering
Track 4: Protein Therapeutics and Market Analysis
Chair
Victor Tsetlin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry-RAS, Russia
Co-Chair
Gamal E. H. Osman
Umm Al-Qura University, Saudi Arabia
Session Introduction
Gamal E. H. Osman
Umm Al-Qura University, Saudi Arabia
Title: Gene Isolation, cloning, nucleotide sequencing and overexpression of anticancer protein from local bacterial isolates
Time : 11:50-12:15
Biography:
Gamal E H Osman has completed his PhD at the age of 34 years from University of Texas at Dallas and Postdoctoral studies from Kansas State University. He is a Professor of Molecular Microbiology at UQU. He has published more than 30 papers in reputed journals.
Abstract:
A total of 407 samples from western region of Saudi Arabia were collected. These samples were collected from both soil samples and dead larvae of Spodoptera littoralis (Lepidoptera) and they were examined for the presence of Bacillus thuringiensis. The bacterium was isolated by acetate-selective enrichment medium and plating. Identification of isolates performed by microscopic examination and analysis of 16S rRNA genes by DNA sequencing for PCR products. The confirmed Bacillus thuringiensis isolates are 22 in total were recovered from 4.6% of soil samples and from 6.6% of dead larvae. Although Bacillus thuringiensis was not found to be abundant in soil habitats in Makkah Province, the results suggest that the bacterium is part of the indigenous microflora of the area we have explored. The 88 kDa parasporin protein was secreted by Bacillus thuringiensis during the stationary phase of growth. Isolated strains were screened for the presence of parasporin genes by Polymerase Chain Reaction (PCR) amplification with only four strains producing the desired bands of parasporin1. The amplified fragments were cloned in pGEM-vector, sequenced and analyzed. The nucleotide sequences of parasporin were given Gene-bank accession numbers: KJ576792 and showed 99% identity with the previously isolated genes in neucleotide level while it was 98% identity in amino acid level. The full length gene was sub-cloned into pET-30a expression vector and overexpressed in E. coli under the control of the inducible T7 promoter. The heterologously produced of parasporin protein (# 30% of total protein) was found in both soluble and insoluble forms. Expressed protein was been purified.
Ing-Ming Chiu
National Health Research Institutes, Taiwan
Title: Neural stem cells promote nerve regeneration through IL12-induced oligodendrocyte differentiation
Time : 12:15-12:40
Biography:
Ing-Ming Chiu has completed his PhD at the age of 29 years from Florida State University and Postdoctoral studies from National Cancer Institute. He is the Director of Division of Regenerative Medicine in Taiwan’s National Health Research Institutes. He has published more than 130 papers in reputed journals and serving as Chief Scientific Officer of Taitheon International Company in Taiwan.
Abstract:
Regeneration of peripheral nerve injury is a slow and complicated process which could be accelerated by implantation of neural stem cells (NSCs) or nerve conduit. We previously developed a novel approach to isolate neuronal progenitor cells from mouse and human brain tissues using F1B-GFP reporter plasmid. We showed that F1B-GFP+ NSCs when combined with FGF1 and nerve conduit could promote the repair of damaged sciatic nerves in mice and rats. Implantation of NSCs combining with conduits promotes the regeneration of damaged nerve may be due to conduit provides support and connection of injured nerve whilst preventing fibrous tissue in growth and retaining neurotrophic factors; implanted NSCs differentiate into Schwann cells and maintain a growth factor-enriched micro-environment which helps nerve fiber regeneration. In this study, we identified IL12p80 (the bioactive homodimer form of IL12p40) in the cell extracts of mice which were implanted with nerve conduit combined NSCs. Levels of IL12p80 in these conduits are 1.89 fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves the motor function recovery and increases the diameter up to 4.5 fold of the regenerated nerve. In vitro study further reveals that IL12p80 stimulates the oligodendrocyte differentiation of mouse NSCs through the phosphorylation of Stat3. These results suggest that IL12p80 can trigger neuroglia differentiation of mouse NSCs through Stat3 phosphorylation and enhance myelination and nerve regeneration process in a mouse sciatic nerve injury model.
Victor Tsetlin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry-RAS, Russia
Title: Neurotoxins: Enemies and/or friends and why protein engineering?
Time : 12:40-13:05
Biography:
Victor Tsetlin has received his PhD in 1973, and since 1996 he is working as a Professor and as a corresponding Member of the Russian Academy of Sciences, Head of the Department since 2006. He was a Visiting Scientist at Imperial College, London in 1985 and Visiting Professor at Free University of Berlin in 1993-1995. He was a Member of ESN Council in 1999-2009, Member of the Advisory Board of the EJB-FEBS Journal in 2000-2011 and from 2013, Member of the Advisory Board at the Biochemical Journal. He is the author of over 250 publications in many journals; among them are Journal of Neurochemistry, Journal of Biological Chemistry, Proceedings of National Academy of Sciences, USA, Nature Structural and Molecular Biology, Trends in Pharmacological Science.
Abstract:
Animal venoms in snakes, spiders, scorpions and other dangerous creatures contain different peptide and protein neurotoxins which on biting cause pain, wounds and often the result is fatal. In this respect the neurotoxins are clearly enemies and the task is to find appropriate antidotes: Most often, it is the production of appropriate antibodies, the process not strictly being in frames of “protein engineeringâ€. However, each venom contain tens and hundreds of different neurotoxic peptides/proteins and due to huge number of species differing in the venom composition, in general the venoms are considered as naturally-occurring peptide/protein libraries of compounds acting mostly on the central nervous system. Thus, it is possible to isolate those which act selectively on a particular enzyme, receptor or ion channel. To-day neurotoxins are invaluable tools in neurobiology and in this respect can be considered as Friends. Protein engineering (in the form of classical chemical modification) is used to identify in those peptides and proteins the functional residues and to prepare labeled derivatives for detecting the desirable receptor targets. Computer modeling of the respective interactions (based on the X-ray or NMR structures of those neurotoxins and their complexes) assists in choosing appropriate modifications in the primary structure and subsequent preparation of the novel peptides and desirable protein mutants by peptide synthesis and heterologous bacterial expression. The described scheme will be illustrated in the lecture by alpha-neurotoxins (proteins) from snake venoms and alpha-conotoxins (peptides) from poisonous Conus mollusks, both interacting with different subtypes of nicotinic acetylcholine receptors.
M Waheed Akhtar
University of the Punjab, Pakistan
Title: Designing antigens for reliable serodiagnosis of tuberculosis
Time : 14:00-14:25
Biography:
M Waheed Akhtar is currently a Professor Emeritus in University of the Punjab, Lahore, Pakistan. His current research interests include engineering cellulases and xylanases and their over-expression to construct a potent enzyme mixture for saccharification of pre-treated plant biomass. His group is also working on designing fusion antigens for a reliable serodiagnosis of tuberculosis. He has supervised research of several dozens of successful PhD graduates and published over 150 research papers.
Abstract:
Antigens of Mycobacterium tuberculosis produce highly variable response in different tuberculosis patients. Thus detection of multiple antibodies is necessary to ensure reliability in serodiagnosis of tuberculosis. Fusion molecules consisting of fragments having epitopes from two or more antigens showing high sensitivity against all the corresponding antibodies would be helpful in achieving this objective. In silico analysis to examine positioning of the epitopes in the fusion molecules can be of great advantage in designing such constructs successfully. We have produced a series of truncated antigens and constructed fusion molecules from epitope regions of several M. tuberculosis antigens like PstS1, TB16.3, echA, HSPX, PE35 and FbpC1. Data obtained on the basis of antibody detection in hundreds of plasma samples of both the smear positive and smear negative tuberculosis patients showed that sensitivity of some of the antigens after truncation increased significantly. Some of the fusion molecules constructed showed sensitivities very similar to the expected combined sensitivity of the contributing antigens. The heat shock protein HSPX of M. tuberculosis not only exhibited full sensitivity but also resulted in soluble expression in E. coli in fusion with some other antigens. Construction of the fusion molecules, their expression at high levels, some in a soluble form and showing high sensitivity in detecting multiple antibodies seem promising for developing a reliable and cost-effective serodiagnosis of tuberculosis.
Jared Cochran
Indiana University, USA
Title: Engineering a “metal switch†into molecular motors to control their activity
Biography:
Jared C Cochran has completed his PhD in 2005 from the Department of Biological Sciences at the University of Pittsburgh and postdoctoral studies in 2011 from the Department of Chemistry at Dartmouth College. He is currently an Assistant Professor in the Department of Molecular and Cellular Biochemistry at Indiana University. He has published 15 papers in reputed journals and has 4 manuscripts in review at present.
Abstract:
Kinesins and myosins are molecular motors that use the energy from nucleotide hydrolysis to carry out cellular tasks. In addition to the P-loop, these proteins use similar structural motifs, called switch-1 and switch-2, to sense and respond to the gamma-phosphate of the nucleotides and coordinate nucleotide hydrolysis. We have developed a strategy to probe metal interactions within kinesins and myosins, by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine (−OH) and cysteine (−SH) amino acids. We present the crystal structure of a recombinant kinesin motor domain bound to MnADP and report on a serine-to-cysteine substitution in the switch-1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn(II) to Mg(II). This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg(II). In multiple kinesin members, this defect is rescued by Mn(II), providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. We also present results for an analogous substitution in non-muscle myosin-2. This mutant myosin shows aberrant actin interaction whereby dissociation becomes rate-limiting in the presence of Mg(II), yet is rescued by Mn(II). There are several relevant and important applications to this metal switch technology that will allow further biophysical characterization of molecular motors and molecular switch proteins.
- 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.
- Young Researchers Forum
Chair
Victor Tsetlin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry-RAS, Russia
Session Introduction
Somnath Mukherjee
University of Chicago, USA
Title: A new high affinity immobilization tag for generating recombinant antibodies by phage display library selection
Time : 14:25-14:45
Biography:
Somnath Mukherjee is currently a Postdoctoral Research Scholar working on protein and antibody engineering in the group of Professor Anthony A Kossiakoff at University of Chicago. He has completed his PhD from Indian Institute of Science and Technology (IIT) Kharagpur, India. He has more than 15 peer reviewed publications in journals of international repute.
Abstract:
Reversible, high affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD ~2 pM), which can be reversed by EDTA allowing controlled “capture and release†of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to over express and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP fused constructs. KD of the Fabs was in sub to low nanomolar (nM) ranges and was successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag, so the fact that all led to successful selection end points means that borderline cases can be worked on with a high probability of positive outcome. Taken together with examples of successful case specific high level applications like generation of conformation, epitope and domain specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well documented drawbacks.
Evan Reynolds
University of North Carolina at Chapel Hill, USA
Title: Superiority through selectivity: Unnatural co-factors and the enzymes that bind them
Time : 14:45-15:05
Biography:
Evan Reynolds is currently a PhD candidate in the Chemistry Department at the University of North Carolina-Chapel Hill working in the lab of Dr. Eric Brustad. His projects are aimed at expanding the chemical functionality available to proteins by developing new techniques in co-factor engineering. He has obtained his BSc with highest distinction in Chemistry and a BA in Mathematics from the University of Virginia; there he studied the photophysical properties of luminescent ruthenium complexes with Dr. James Demas. He has published a report in the Journal of Fluorescence describing viscosity effects on rates of oxygen quenching of these luminescent complexes.
Abstract:
Nature uses co-factors to expand the chemical functionality of proteins beyond that of the amino acids. Heme is an especially versatile co-factor in nature having functions in oxygen transport, cell signaling and oxidation catalysis. In all of these roles, the heme co-factor supplies activity while the protein environment tunes selectivity towards a specific purpose. This concept has been utilized by protein engineers to tune the protein environment towards a specific application while maintaining the activity provided by the heme co-factor. In this way, heme proteins have been engineered as catalysts for unnatural reactions such as cyclopropanation and also as useful contrast agents in Magnetic Resonance Imaging (MRI) for detection of neurotransmitters in the brain. Although the heme co-factor provides the activity for these applications, it also limits how far we can go in utilizing enzymes for these purposes. The goal of my project is to develop unnatural heme derivatives that expand the chemistry of these enzymes even further. By engineering proteins that selectively bind and utilize unnatural heme co-factors, we can efficiently introduce new activity to proteins in vivo. Towards, this goal I have developed a series of synthetic heme derivatives with an altered porphyrin scaffold and or different metal center. These synthetic modifications allow the properties of the co-factor to be tuned and also serve as a handle around which we can design the enzyme for selective binding of the synthetic co-factor. The synthetic co-factors I have developed display improved activity relative to heme in unnatural cyclopropanation reactions.
Samrat Roy Choudhury
Purdue University, USA
Title: Optogenetic control of endogenous neuronal commitment and differentiation through epigenetic amendments of Ascl1 (Mash1) promoter
Time : 15:05-15:25
Biography:
Samrat Roy Choudhury has graduated in Nanobiotechnology from the Biological Sciences Division of Indian Statistical Institute, India. He is currently pursuing his Postdoctoral Research at the Purdue University, USA. He has received several prestigious Doctoral and Postdoctoral Fellowships from the Indian Government. He has more that 20 peer reviewed publications in the international journals, patients, books and book chapters to his credit.
Abstract:
Achaete-Scute homolog 1 (Ascl1 or Mash1 in mammal) is an important candidate of proneural genes, known to promote cell cycle exit and neuronal differentiation. Mash1 initiates the neuroblast differentiation from neuroepithelial cells and also protect the neuroblasts from damages through ‘delta’ protein mediated lateral inhibition machinery during nervous system development. Aberrant methylation status at the proneural gene promoters however may lead to their ectopic expressions which have been recognized in conjunction with impaired nervous growth, increase of excitatory neurons or acute neuralgia. Herein, we have targeted intelligently engineered light inducible (optogenetic) fusion protein tools to demethylate the Mash1 promoter with spatiotemporal precision, which otherwise identified hypermethylated with reduced expression in a few murine neural stem cell (NSC) lineages. The promoter targeting construct contained blue light inducible protein CIB1 (cryptochrome-interacting basic helix loop helix) fused to the Ascl1 promoter specific transcription activator-like effectors (TAL-TFs), while the CIB1 interacting protein partner CRY2 was fused to the ten-eleven translocation proteins (TET). Light induced association of these optogenetic fusion proteins resulted in significant selective demethylation at the target CpGs of Mash1 promoter with increased gene expression. The overall outcome of these light induced epigenetic changes was then analyzed in regard to the altered phenotype and fashion of differentiation amongst the NSCs. We also introduced several single molecule fluorescence tools like FLIM-FRET or FCS to monitor intra-nuclear association rate and binding dynamics of the optogenetic proteins. This system hence, allows direct and non-invasive probing of the critical stages of NSC morphogenesis through light induced epigenetic alterations and transcriptional activation.
J Madhumathi
Indian Institute of Technology, India
Title: Recombinant fusion proteins for targeted therapy of leukemia
Time : 15:45-16:05
Biography:
Madhumathi J completed her PhD at Anna University, Chennai which was on the development of multi-epitope peptide vaccines for Lymphatic Filariasis. She has 17 publications, two patents for filarial vaccines, two Genbank submissions and a protein structure submission in PDB. She received New Investigator award from the International Society of Infectious Diseases, USA on April 2014 for vaccine study. Currently, her Post-doctoral work in Indian Institute of Technology, Chennai involves identifying cancer stem cells in leukemia and targeting them. She received the young scientist grant to carry out this project from the Department of Science and Technology, India.
Abstract:
Recombinant immunotoxins are antibody-toxin chimeric molecules that kill cancer cells via binding to a surface antigen, internalization and delivery of the toxin moiety to the cell cytosol. Immunotoxins target the surface of cancer cells with considerable potency, using protein toxins capable of killing a cell with a single molecule. It is well established that cancer cells overexpress several tumor associated antigens, membrane receptors, and carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments (scFv) targeted against these antigens are fused with bacterial or plant toxins and used as immunotoxins. The recent emergence of a new class of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein has reduced immunogenicity and toxicity. We have developed humanized chimeric toxins using human TRAIL and IL2, which specifically targets leukemic cells leaving normal cells. The two most important cytokines used as targeting molecules with anti-tumor activity approved by FDA for cancer treatment are IL2 and type I IFN. IL2 promotes natural killer cells, T cells and acts as a mitogen and interferes with blood flow to tumor. Low dose recombinant IL2 is proved to activate antitumor immune response in advanced malignancies. Soluble recombinant TRAIL (rTRAIL) induces apoptotic cell death in a wide variety of tumor cell lines in vitro, while sparing most normal cells. Importantly, no apparent systemic toxicity of rTRAIL was also observed in non-human primates. Humanized immunotoxins using IL-2 as the target ligand and TRAIL as the toxin moiety would highly improve the therapeutic efficacy in cancer.
Modupe Ajayi
University of Leeds, UK
Title: Identification of high affinity Adhirons for the development of rapid point-of-care diagnostics for Clostridium difficile infection
Time : 16:05-16:25
Biography:
Modupe Ajayi is a third year PhD student at the University of Leeds, United Kingdom. She is a recipient of several national and international scholarships including that for her PhD. She is driven by the passion to use protein engineering as a tool for improving the quality of life.
Abstract:
Clostridium difficile (C. diff) is a leading cause of hospital acquired infection, and antibiotic-associated diarrhoea. Point-of-care tests would be valuable for rapid diagnosis of patient with C. diff infection both in hospital and community settings. Non-antibody binding proteins are increasingly being used as alternatives to antibodies and we have developed a very stable (Tm=101oC) non-antibody binding protein called Adhiron (commercialized by Avacta Life Sciences Ltd as Affimer Type II). High quality phage display libraries have been used to identify Adhirons against >200 targets. These have potential applications including as scientific research reagents, in diagnostics, imaging, therapeutics and drug discovery. We have identified a number of specific and non-cross-reactive binders against the three well established biomarkers of C. diff infection, glutamate dehydrogenase, toxin A and toxin B. The characterization of these Adhirons and their use in developing a point-of-care diagnostic tool for C. diff infection will be presented.
Biography:
Nasir Ali has completed his PhD from Xiamen University. He is born in 1987 at KP province Pakistan. He has completed his Master’s degree from University of Peshawar. He has published more than 5 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
The cDNA gene (AnBgL1), encoding GH3 family β-glucosidase (EC3.2.1.21) from Aspergillus niger BE-2 (abbreviated to AnBgL1) was amplified and inserted into the yeast expression pPIC9K vector at the site of Bln I (Avr II) and Not1. The recombinant expression vector designated as pPIC9K-AnBgL1 was transformed into Pichia pastoris GS115. The transformants were screened on a MD plate which inoculated on geneticin G418-containing YPD plates. The transformants expressed the high β-glucosidase activity of 22.6 U/ml. SDS-PAGE assay demonstrated that the AnBgL1 was extracellularly expressed with an apparent M.W. of 90.0 kDa. The purified AnBgL1 displayed the maximum activity at pH 6.0 and 60° C. It was highly stable at a broad pH range of 4.0-7.5, and at a temperature of 60° C. The Km and Vmax, towards p-NPG at pH 5.5 and 60° C were 1.45 mg/ml and 2,365 U/mg, respectively. The AnBgL1 displays high similarity to the β-glucosidases of A. niger (FN430671) and A. niger (DQ655704), the members of the GH3 family. The β-glucosidase gene (Bgl1) from A. niger was cloned and recombined with cbh1 optimized promoter (pcbh1) and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1 and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT) using hygromycin B resistance gene as the screening marker. Bgl-1 transformants was screened. The enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformants increased by 8.5% and 15.2% under induction condition, respectively compared with the host strain EU7-22. The results showed that the cbh1 promoter (pcbh1) has successfully driven the over-expression of Bgl1 gene in T. orientalis under glucose repression condition.
Hyun Joon Chang
Korea University, Korea
Title: The mechanical impact of Aromatic residue mutation on Aβ amyloid protofibrils
Time : 16:45-17:05
Biography:
Hyun Joon Chang has completed his Bachelor’s degree from Korea University and he is currently pursuing his Doctoral studies in Korea University, Deparment of Mechanical Engineering. He is majoring in Protein Engineering especially in computational protein engineering using Molecular Dynamics. He has published 2 papers in reputed journals and he is currently a Member of Global PhD Fellowship funded by Korea Research Foundation.
Abstract:
Amyloid proteins are the main cause of neuro-degenerative and degenerative diseases such as Alzheimer’s disease, Parkinson’s disease and so on. These proteins self-assemble due to their physiological conditions, e.g., temperature, pH and internal fluctuation. They are known to be structurally stable due to their residues’ intermolecular forces, hydrogen bond for example. Recently, the aromatic residues, phenylalanine residue to exemplify have been recognized to serve as a stability source of amyloid fibrils. Yoon et al. revealed the structural stability of hIAPP fibril with a partial mutation from phenylalanine residue to leucine residue, announcing that the wild-type models possess larger structural properties and reaction forces than the mutated models. In addition, experimental study of Aβ amyloid fibrils with aromatic residue mutation was recently conducted to reveal the aggregation and formation tendencies of the amyloid fibrils. In this study, we further investigate the structural stability and properties of Aβ fibrils at atomic scale using Molecular Dynamics (MD) simulations. We reveal the role of the aromatic residue mutation effect on the Aβ fibrils through the material properties and observe the specific interaction between phenylalanine and leucine residue which affects the overall structural properties and stabilities. This study may serve as a foundation for target treatment strategy of neuro-degenerative diseases in near future.
Atsbeha Gebreegziabxier
Ethiopian Public Health Institute, Ethiopia
Title: Co-immobilization by DNA binding protein tags
Time : 17:05-17:25
Biography:
Atsbeha has completed his MSc at the age of 27 years from Universitat Rovira I Virgili, Spain. He is the director of HIV/AIDS Research, EPHI, Ethiopia. He has published more than 4 papers in reputed journals.
Abstract:
The development of improved protein immobilization approaches is a significant step for many biotechnological applications. A large array of different protein immobilization approaches have been developed based on physical, covalent and bioaffinity interactions. Most of these immobilization techniques only allow for the immobilization on the surface of a single target protein and do not allow the controlled co-immobilization of several proteins. Therefore, we aspire to develop a system that allows controlling the structure of a multiple protein complex both in solution and on surfaces. To do this we propose to use several DNA binding proteins with different sequence specificities and high binding affinities as fusion tags to the target proteins to be immobilized. In this system, the co-immobilization of the target proteins is controlled by the localization of specific sequences on a double stranded DNA molecule. In this work, we performed experiments as a proof of concept for the proposed novel immobilization system based on DNA binding proteins tags. Specifically, two different DNA binding proteins were selected (scCro16 and SpoIIID) as candidates for the role of DNA binding protein tags. These proteins were successfully expressed in E. coli and purified using ion exchange chromatography and we optimized and performed an Electrophoretic Mobility Shift Assay (EMSA) to assess the suitability of the selected DNA binding proteins to work as DNA binding tags in the context of the proposed immobilization system. The EMSA assay showed that scCro16 and SpoIIID works as expected binding to its specific DNA binding sequence
Zain Ullah
Gomal University, Pakistan
Title: Analysis of biophysical studies of metal binding to zinc α2 glycoprotein (ZAG) using fluorescence
Biography:
Zain Ullah has completed his MPhil degree from Department of Chemistry, University of Kohat (KUST) and is now a regular student of PhD at last stage in Department of Chemistry, Gomal University. He has published 10 papers in international reputed journals.
Abstract:
Zinc-Alpha-2-Glycoprotein (ZAG), is present in blood, sweat, seminal fluid, breast cyst fluid, serum, saliva, cerebrospinal fluid, milk, urine, and amniotic fluid (1)(2). As the protein’s prominence may suggest, there are a range of metabolic functions associated with this protein including lipid lipolysis. ZAG functions in lipid lipolysis by binding fatty acids from triglycerides and decreasing levels of stored fats, resulting in body fat loss. The function of ZAG under physiologic and cancerous conditions remains mysterious but is considered as a tumor biomarker for various carcinomas. ZAG was first isolated from human blood plasma via precipitation using 20 mM Zinc Acetate. It showed similar electrophoretic mobility to α1 immunoglobin and hence were named Zinc Alpha 2 glycoprotein. To my best of our knowledge, no studies have been directly examined to metal binding by ZAG. Preliminary studies in the McDermott laboratory suggest that ZAG is able to bind zinc. This study will examine binding of metals from the IRVINE WILLIAMS series to ZAG using fluorescence.