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IMTECH, Chandigarh APPLIED TO DOMAIN EXPERTISE • CLONING & EXPRESSION OF RECOMBINANT PROTEINS • PROTEIN/ENZYME FUNCTION • PROTEIN FOLDING/ MISFOLDING + Bioinformatics Immunology Monoclonal Abs Animal Handling Cell culture Yeast Genetics PROTEIN / ENZYME SCIENCE & TECHNOLOGY Structures from IMTECH Representative recent publications •Ultra-high yielding Process for recombinant Streptokinase Production. Hydantoinase Radha Kishan & coworkers J.Mol.Biol. 347, 95-105 (2003) A backbone-reversed all-beta polypeptide (retro-CspA) folds and assembles into amyloid nanofibres. Protein Engineering 16, 875-879. - Under clinical trials [Shasun Drugs and Chemicals Ltd.] •Process for Engineered, Improved “clot-specific” Streptokinase - Technology transferred [Nostrum Pharmaceuticals, USA] (2004) Folding of beta/alpha-unit scrambled forms of S. cerevisiae triosephosphate isomerase: Evidence for autonomy of substructure formation and plasticity of hydrophobic and hydrogen bonding interactions in the core of the (beta/alpha)8-barrel. Proteins : Structure, Function & Bioinformatics 55, 548-557. •Design and make generics and second-generation generics •Develop therapeutic antibodies •Develop transgenic technologies for protein therapeutic generation •Develop novel expression systems (e.g., performing glycosylation) •Process for recombinant Staphylokinase - Technology transferred [Strides Arcolab Ltd] Eps8 SH3 Radha Kishan & coworkers Protein Sci. 10, 1046-1055 GroES Mande & coworkers Protein Eng. 14, 391-395 RESEARCH AREAS Folding , stabilization & engineering of natural /designer proteins. Amyloidogenesis Structure/Function/Engineering of therapeutic proteins (streptokinase/growth hormone) Structural constrainment of synthetic peptides Structural biology of signal transduction / SH3 domains •Processes for three recombinant animal growth hormones - Packages ready for technology transfer Exploiting enzymes through engineering of structure / folding / stability / activity * New engineered cellulases, proteases, amylases, lipases (R.T activity, high stability) * Biofuels from plant biomass Structural biology of proteins from pathogenic organisms & hyperthermostable proteins Microbial factories for the production / engineering of therapeutic proteins IMPROVED FUNCTION - Now commercialized [Cadila Pharma Ltd.] (2003) A backbone-reversed form of an all-beta alpha-crystallin domain from a small heat-shock protein (retro-HSP12.6) folds and assembles into structured multimers. Journal of Biological Chemistry. 278, 26505-26510. Adding value to therapeutic proteins through engineering of structure / folding / stability / activity • ENGINEERING TOWARDS •Complete high-efficiency process package for the production of Natural Streptokinase (2003) Domain truncation studies reveal that the Streptokinase-Plasmin activator complex utilizes long-range protein-protein Interactions with macromolecular substrate to maximize catalytic turn-over. Journal of Biological Chemistry: 278: 30569 (2005) Molecular Structure of D-Hydantoinase From Bacillus Sp. AR9: Evidence for Mercury Inhibition. Journal of Molecular Biology. 347:95-105. 2005. & MODELLING Representative Protein Technologies (2002) Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the streptokinase-plasminogen activator complex through interaction with substrate kringle domains. Journal of Biological Chemistry 277: 13257. (2004) Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase of Mycobacterium Tuberculosis. Journal of Biological Chemistry. 279:43595-43603. 2004. • STRUCTURE DETERMINATION Available Technologies for Enzymes DONE Alkaline Protease 1 – Good for blood stain removals * New processes for textile / food / leather industries Technologies under development – Hyperthermophile enzymes from archaea and bacteria Technology for production of natural streptokinase from Streptococcus sp. Hyperthermophilic archaea constitute the third domain of life on earth Alkaline Protease 2 – Good for alkaline applications Technology for production of recombinant streptokinase -Amylase (recombinant) Technology for an engineered clot-specific streptokinase Second-generation (engineered) IL-2, IFN-G, EPO Thermostable endo-xylanase (recombinant) Technology for production of recombinant staphylokinase Thermostable endo-glucanase (recombinant) Therapeutic antibodies DOING They live in magma-heated springs, geysers and deep-sea hydrothermal magma vents at temperatures of 80-120 degrees Centigrade Technology for production of recombinant growth hormones Capillary Electrophoresis MALDI-TOF MS Stopped-Flow CD & Fluorescence Amino Acid Analysis