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.