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Introduction Proteins Major functional molecules • Consist of linear polymers built from series of up to 20 different Lamino acids • Play central roles in biological events through interactions with cognate proteins / ligands - Cell signaling processes • Enzymes catalyze the reactions with high specificity and catalytic efficiency in cellular metabolic pathways : major components Practical use • Therapeutic agents - Many diseases caused by mutations and loss of their functions - Cancers, lysosomal storage disorders, Alzheimer's disease etc. • Industrial Biotechnology: Use of enzymes for bio-based processes • Biomaterials: Biocompatible materials Protein-protein interaction network Basic components Peptide bond Torsion or dihedral angles Planarity results from the delocalization of the lone pair of electrons of the nitrogen onto the carbonyl oxygen Conformation of the polypeptide backbone : - location in space of the three sets of atoms that are linked together, namely the alpha carbon, carboxyl carbon, and amide nitrogen atoms - Secondary structure is defined by the rotation of the planar peptide units around the bonds connecting the alpha carbon atoms - Number of conformations available to the polypeptide chains are restricted by steric clashes -Conformational space in a two-dimensional plot of ψ and ϕ :Ramachandran diagram Protein structure • Primary structure: amino acid sequence • Secondary structure: Polypeptides are organized into hydrogenbonded structures regularly repeating local structures stabilized by hydrogen bonds. Alpha helix, beta sheet and turns • Tertiary structure: the overall shape of a single protein molecule • Quaternary structure: the structure formed by several subunits 3D structure of the protein myoglobin showing turquoise alpha helices. This protein was the first to have its structure solved by X-ray crystallography in 1958. Towards the right-center among the coils, a prosthetic group called a heme group (shown in gray) with a bound oxygen molecule (red). PDB database Experimental Method Proteins Nucleic acids Protein/ Nucleic Acid complexes Other Total X-ray 92427 1645 4600 4 98676 NMR 9686 1124 227 8 11045 584 29 191 0 804 Hybrid 70 3 2 1 76 Other 166 4 6 13 189 102933 2805 5026 26 110790 EM Total: August 2015 Examples of protein structures from PDB Protein Engineering Method to develop more useful or valuable proteins • Alteration of a single amino acid residues at specific site • Insertion or deletion of a single amino acid residue • Alteration or deletion of a segment or an entire domain • Generation of a novel fusion protein • Incorporation of unnatural amino acids at specific site Why Protein Engineering ? • Proteins/Enzymes : Evolved for host itself, not for human - Most proficient catalysts with high specificity • Need further improvement for practical use: - Specificity : Cognate ligands, substrates - Binding affinity - Stability - Catalytic activity - Folding/Expression level etc.. • Goal of protein engineering : - Design of protein/enzyme with desired function and property for practical applications Designer proteins/Enzymes Ex) Therapeutic proteins, Industrial enzymes, Fluorescent proteins, Protein binders Technology Development Random approach Structure-based rational approach - Screening from nature - Random mutations - Evolutionary approach - Directed evolution • • • • Accumulation of beneficial mutations No structural data HTS system Construction of diverse library Structure-function relationship Site-directed/saturation mutagenesis Computational (in silico) method - Virtual screening of large sequence space - Large structural data : >~30,000 - High computing power - Mechanistic knowledge Combinatorial approach - Structure-based design - Evolutionary method - Computational method Biomolecular Eng. Lab. New version of therapeutic proteins by Protein Eng • EPO (Erythropoietin) with a longer plasma half-life by incorporation of additional N-glycosylation • Faster-acting insulin by modification of amino acid sequence • Slow-acting insulin • Faster-acting tissue plasminogen activator(t-PA) by removal of three of the five native domains higher clot-degrading activity • Ontak : A fusion protein consisting of the diphtheria toxin linked to IL-2 Selectively kills cells expressing the IL-2 receptor Approved for the treatment of cutaneous T cell lymphoma in 1999 in US • Bi-specific monoclonal antibodies for dual targets higher efficacy Glycoprotein: Glycosylation, Glycobiology • Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains • One of the most important post-translational modifications (PTMs) : N-glycosylation / O-glycosylation in Mammalian / Yeast • Essential roles in in vivo : Biological activity, folding, solubility, protease resistance, immunogenicity, signal transduction, and pharmacokinetics • Carbohydrates on cell surface : Cell signaling, cell attachment, cell adhesion, recognition, and inflammation • About 60 % of therapeutic proteins are glycoprotein - Therapeutic proteins : 140 approved - EPO Glycan Profile of Glycoprotein Man Man ASN GlcNAc GlcNAc Gal ASN GlcNAc Man Man Human (in Golgi) Man NANA GlcNAc Gal GlcNAc GlcNAc NANA Pichia pastoris (in Golgi) Man Man Man Man Man Man Man Man Man Man Man Man • Glycan profile: very complex and varies broadly, depending on cell types and production conditions : Glycan moiety, occupation number, length of glycosylation chain • Therapeutic proteins require proper glycosylation for biological efficacy → Analysis of glycan profile, its role / function in vivo, glycosylation pathway, and property of glycoproteins are a key to Glycobiology Monosaccharide structures Fucose, Fuc C6O5H12 Sialic Acid, NAcNeuA C11O9NH19 Galactose, Gal C6O6H12 Mannose, Man C6O6H12 N-Acetylgalactosamine, GalNAc C8O6NH15 N-Acetylglucosamine, GlcNAc C8O6NH15 N-Linked glycan structures Example of tetraantennary complex glycan that contains terminal sialic acid residues, a bisecting GlcNAc on the pentasaccharide core, and fucosylation on the core GlcNAc. Erythropoietin (EPO) • Glycoprotein : Growth factor (166 amino acids, MW 34 kDa) produced in kidney Promote the formation of red blood cells(erythrocytes) in the bone marrow • Binds to the erythropoietin receptor on the red cell progenitor surface and activates a JAK2 signaling cascade • Clinically used in treating anemia resulting from chronic kidney disease, inflammatory bowel disease (Crohn's disease and ulcer colitis), and myelodysplasia from the treatment of cancer (chemotherapy and radiation) • Carbohydrate moiety : in vivo activity, stability, solubility, cellular processing and secretion, immunogenicity • Three N-glycosylation sites and one O-glycosylation site • About 50 % of EPO’ secondary structure : α-Helix • Carbohydrate content : ~ 40 % Glycosylation pattern of EPO History of the EPO development • 1971: First purified from the plasma of anemic sheep • 1985 : Produced by recombinant DNA technology • 1989: Approved by FDA for treatment of anemia resulting from chronic kidney disease and cancer treatment (chemotherapy and radiation) • Total sales : $ 11 billion (2005) • Major EPO brands : Biosimilars - Epogen by Amgen ($ 2.5 billion) - Procrit by Ortho Biotech ($ 3.5 billion) - Neorecormon by Boehringer-Mannheim ($ 1.5 billion) New version of EPO by protein engineering • As the patent becomes expired, Amgen wanted to prolong the market share by developing a new version of EPO by protein engineering • Aranesp : Introduction of two additional N-glycosylation sites - Which site of EPO? A prolonged serum half-life from 4-6 up to 21 hrs - What benefit to patients? Launched in 2001 Current sale : $ 3.5 billion Design of hyper-glycosylated EPO • Relationship between sialic acid content, in vivo activity, and serum half-life • Hypothesis: Increasing the sialic acid-containing carbohydrate of EPO increase in serum half-life in vivo biological efficacy Design procedure • N-linked carbohydrate is attached to the polypeptide backbone at a consens us sequence for carbohydrate addition: Asn-Xxx-Ser/Thr -The middle amino acid can not be proline (Pro). • Critical factors: - Local protein folding and conformation during biosynthesis: Co-translation - No interference with receptor binding - Stability • Structure-based engineering: site-directed mutagenesis - Effect on bioactivity and conformation: Structure/function relationship - Identification of the residues critical for EPO receptor interaction and proper folding of EPO - Generation of EPO analogues with amino acid change at five positions Ala30ASn, His32Thr, Pro87Thr, Trp88Asn, Pro90Thr - Two additional carbohydrate sites at positions 30 and 88: Aranesp Development of enzyme process: Atorvastatin (Lipitor) • Lipitor : Brand name by Pfizer • A competitive inhibitor of HMG -CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase) - HMG-CoA reductase catalyzes the reduction of 3-hydroxy-3-methylglutarylcoenzymeA (HMG-CoA) to mevalonate, which is the rate-limiting step in hepatic cholesterol biosynthesis. • Inhibition of the enzyme decreases de novo cholesterol synthesis, increasing expression of low-density lipoprotein receptors (L-receptors) on hepatocytes. Increase in LDL uptake by the hepatocytes, decreasing the amount of LDL-cholesterol in the blood. • Like other statins, atorvastatin also reduces blood levels of triglycerides and slightly increases the levels of HDL-cholesterol. • The largest selling drug in the world : $ 12.9 billion in 2006 • Generic drug : Simvastatin by Merck Lipitor Pfizer fight against a simvastatin generic • Doctors and patients began switching to a cheaper generic alternative drug called simvastatin from Merck. • Pfizer launched a campaign including advertisements, lobbying efforts, and a paid speaking tour by Dr. Louis W. Sullivan, a former secretary of the federal Depart. of Health and Human Services, to discourage the trend. • Studies show that at commonly prescribed doses Lipitor and simvastatin are equally effective at reducing LDL cholesterol. • Pfizer has begun promoting a study, conducted by Pfizer’s own researchers, concluding switching increased the rate of heart attacks among British patients. Economic process using an enzyme with higher efficiency Halohydrin dehalogenase (HHDH) • Catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, yielding an epoxide • Interconverts halohydrins and epoxides Manufacture of ethyl (R)-4-cyano-3-hydroxybutyrate (HN) from ethyl ethyl (S) -4 –chloro-3-hydroxybutyrate(ECHB) • NH: Starting material for the production of the cholesterol-lowering drug : Atorvastatin (Lipitor) • The specifications for the chemical and enantio-purity of HN are tightly controlled. The hydroxyl in HN is defined the second stereo-center in atorovastatin, and high chemical purity is essential for downstream chemistry Essential for more economic process Halohydrin and Epoxide • A type of organic compound or functional group in which one carbon atom has a halogen substituent, and an adjacent carbon atom has a hydroxyl substituent. • An epoxide is a cyclic ether with three ring atoms General structure of a halohydrin, where X = I, Br, F, or Cl Epoxide Reaction scheme catalyzed by Halohydrin dehalogenase HCN Tetramer Monomer Design criteria • Enzyme source - Expression of HHDH from Agrobacterium radiobacter in E. coli - Volumetric productivity : 6 X 10 -3 g product/L/hour/ gram of biocatalyst • Requirement for implementing the enzyme process at commercial scale - Yield : Complete conversion (100 %) of at least 100 g per liter substrate - Volumetric productivity : > 20 g product /liter/hour/gram of biocatalyst Nature Biotech, 25, 338-344 (2007) Agrobacterium radiobacter halohydrin dehalogenase with its substrate (white). Integration of computational analysis with experimental screening to identify 37 mutations (yellow) that increase enzymatic activity ~4,000-fold Increase in the volumetric productivity : ~ 4,000 fold Development of a more economic process Structural and functional features of Immunoglobulin Ab 2 CDR 2 1 3 3 1 FR VL VH Engineering of Ab for therapeutics • Reduced immunogenicity : Humanization, Human Ab • Improved affinity : Engineering of variable domains ( < nM) • Antibody fragment : Fab, single-chain Fv (scFv), minibody, diabody • Novel effector function: Conjugation with radioisotope, cytotoxic drug • Improved effector function : Fc engineering • Longer half-life: Fc engineering (FcRn binding site) • Bi-specific antibody Engineering of Ab for humanization Antibody Fragments • Better tissue penetration, fast clearance, no effector function • Local delivery • Production in bacterial system Lucentis • A monoclonal antibody fragment (Fab) derived from the same parent mouse antibody as Avastin • Much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF-A • An anti-angiogenic protein to treat the "wet" type of age-related macular degeneration (AMD, also ARMD), Common form of an age-related vision loss. • • Cost $1,593 per dose, compared to Avastin that cost $42. Developed by Genentech and is marketed in the United States by Genentech and elsewhere by Novartis Intermediate age-related macular degeneration Top 8 blockbuster biologicals (2013) Brand name Active ingredient Type Class Humira adalimumab Antibody Remicade infliximab Rituxan 2013 global sales Patent expiry (US$ billion) EU/US Treatment Company TNF inhibitor Arthritis Abbott/Eisai 10.7 Apr 2018/ Dec 2016 Antibody TNF inhibitor Arthritis Merck/Mitsubishi 8.9 Aug 2014/ Sep 2018 rituximab Antibody Anti-CD20 Arthritis, NHL Roche/Biogen 8.6 Nov 2013/ Dec 2018 Enbrel etanercept Antibody TNF inhibitor Arthritis Amgen/Pfizer 8.3 Feb 2015/ Nov 2028 Lantus insulin glargine Protein Insulin receptor Diabetes Sanofi 7.8 2014/2014 Avastin bevacizumab Antibody Anti-angiogenesis Cancer Roche 7.0 Jan 2022/ Jul 2019 Herceptin trastuzumab Antibody Anti-HER2 Breast cancer Roche 6.8 Jul 2014/ Jun 2019 Neulasta pegfilgrastim Protein G-CSF Neutropenia Amgen 4.4 Aug 2017/ Oct 2015 Fluorescent proteins History of Fluorescent Proteins • 1960s : Curiosity about what made the jellyfish Aequorea victoria glow Green protein was purified from jellyfish by Osamu Shimomura in Japan. • Its utility as a tool for molecular biologists was not realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wt-GFP in Gene. - The funding for this project had run out, and Prasher sent cDNA samples to several labs. • 1994 : Expression of the coding sequence of fluorescent GFP in heterologous cells of E. Coli and C. elegans by the lab of Martin Chalfie : publication in Science. • Although this wt-GFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, poor photo-stability, and poor folding at 37°C. • 1996 : Crystal structure of a GFP Providing vital background on chromophore formation and neighboring residue interactions. Researchers have modified these residues using protein engineering (site directed and random mutagenesis) Generation of a wide variety of GFP derivatives emitting different colors ; CFP, YFP, CFP by Roger Y. Tsien group ex) Single point mutation (S65T) reported in Nature (1995) - This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. Applications in many areas including cell biology, drug discovery, diagnostics, genetics, etc. • 2008 : Martin Chalfie, Osamu Shimomura and Roger Y. Tsien shared the Nobel Prize in Chemistry for their discovery and development of the fluorescent proteins. Fluorescent proteins • Revolutionized medical and biological sciences by providing a way to monitor how individual genes are regulated and expressed within a living cell ; Localization and tracing of a target protein in the cells • Widespread use by their expression in other organisms as a reporter usually fused to N- or C terminus of proteins by gene manipulation • Key internal residues are modified during maturation to form the p-hydroxybenzylideneimidazolinon chromophore, located in the central helix and surrounded by 11 ß-strands (ß-can structure) • GFP variants : BFP, CFP, YFP • Red fluorescent protein from coral reef : tetrameric, slow maturation - Monomeric RFP by protein engineering • Quantum yield : 0.17 (BFP) ~ 0.79 (GFP) GFP (Green Fluorescent Protein) • Jellyfish Aequorea victoria • A tightly packed -can (11 -sheets) enclosing an -helix containing the chromophore • 238 amino acids • Chromophore – Cyclic tripeptide derived from Ser(65)-Tyr(66)-Gly(67) • Wt-GFP absorbs UV and blue light (395nm and 470nm) and emits green light (maximally at 509nm) Chromophore formation in GFP GFP and chromophore - Covalently bonded chromophore : 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI). HBI is nonfluorescent in the absence of the properly folded GFP scaffold and exists mainly in the unionized phenol form in wt-GFP. - Maturation (post-translational modification) : Inward-facing side chains of the barrel induce specific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that induce ionization of HBI to the phenolate form and chromophore formation. - The hydrogen-bonding network and electron-stacking interactions with these side chains influence the color, intensity and photo-stability of GFP and its numerous derivatives Diverse Fluorescent Proteins by Protein Engineering wtGFP : Ser(65)-Tyr(66)-Gly(67) Fluorescence emission by diverse fluorescent Proteins The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins. Absorption and emission spectra a) Normalized absorption and b) Fluorescence profiles of representative fluorescent proteins: cyan fluorescent protein (cyan), GFP, Zs Green, yellow fluorescent protein (YFP), and three variants of red fluorescent protein (DS Red2, AS Red2, HC Red). From Clontech.