Day 2 :
Keynote Forum
Victor Tsetlin
Russian Academy of Sciences, Russia
Keynote: From peptide and protein neurotoxins to receptor structure-function and new drugs
Time : 10:00-10:45
Biography:
Victor Tsetlin has received his PhD in 1973 from 1996 Professor, from 2006 – Corresponding Member of the Russian Academy of Sciences, Head of the Department.
Abstract:
Alpha-Neurotoxins, snake venoms proteins, earlier helped to isolate nicotinic acetylcholine receptors (nAChRs) and are still precious tools in their research. The nAChRs are involved in muscle contraction, cognition, immune system activity. nAChR malfunctioning is associated with muscle dystrophies, psychiatric and neurodegenerative diseases, cancer. Another tool in nAChR research is α-conotoxins, neurotoxic peptides from Conus snails. Analysis of nAChR interactions with α-neurotoxins and α-conotoxins provided information about binding surfaces required for drug design. The lecture will cover research at our department in collaboration with European laboratories. First X-ray structure for α-conotoxin complexes with their biological targets was for α-conotoxin PnIA [A10, K14] bound to acetylcholine-binding protein (AChBP); recently from this α-conotoxin several more potent and selective analogs were prepared. Dimeric α-cobratoxin was discovered wherein two α-cobratoxins are joined by 2 intermolecular S-S-bonds; this post-translational modification retained blocking of α7 and muscle-type nAChRs, but added inhibition of α3β2 nAChRs; X-ray analysis revealed unusual packing. α-Bungarotoxin and α-cobratoxin, two closely related α-neurotoxins, block α7 and muscle-type nAChRs with similar affinity; recently we demonstrated that they also block GABA-A receptors, some receptor subtypes being more sensitive to α-cobratoxin. Structurally, α-neurotoxins resemble some mammalian proteins of Ly6 family which modulate the nAChR activity; water-soluble analog of Lynx1 bound competitively to AChBP and muscle-type nAChRs, but non-competitively to neuronal ones.
Keynote Forum
Karl Freed
University of Chicago, USA
Keynote: Protein folding without use of homology or machine learning
Time : 10:45-11:30
Biography:
Karl F Freed has received his BS in Chemical Engineering from Columbia University 1963) and a MA (1965) in Physics and a PhD (1967) in Chemical Physics from Harvard University with Postdoctoral studies in Theoretical Physics at the University of Manchester, England before joining the University of Chicago (1968) where he is the Henry G Gale Distinguished Service Professor, Emeritus. He has published more than 600 papers and he has received the ACS Award in Pure Chemistry and the APS Polymer Physics Prize and he is a Fellow of the American Academy of Arts and Sciences.
Abstract:
Theoretical descriptions of protein folding centers on predicting the folded structure i.e., the relation between sequence and structure and the folding pathway relying heavily on machine learning techniques for predicting the secondary structure and for relating the known native structure to the folding pathway. However, despite their obvious utility, these methods encounter roadblocks hindering the description of proteins with unknown structure and poor homology including many examples in pathogens as well as for proteins with large inserts and deletions. Developing a theory of protein structure and folding pathways without the use of machine learning or homology focuses attention on deducing the fundamental principles of folding, principles that will be essential in devising theories to guide in designing unique proteins and for dealing with more complex processes such as protein-protein recognition. We have developed a de novo approach to protein folding based on the principle of the sequential stabilization of elements of secondary structure. Using a reduced model in which the only degrees of freedom are the backbone dihedral angles only and beginning only with a knowledge of the sequence, a series of rounds of very rapid Monte Carlo simulated annealing trajectories are run to uncover the correlations exhibited by the trajectories in a given round. The correlations then provide biases on the possible backbone conformations in subsequent rounds of trajectories. The convergence of this procedure yields a self-consistent determination of the secondary and tertiary structure as well as the folding pathway.
- 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.
- 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.