1. Introduction

2. Proteins Consist of linear polymers built from series of up to 20 different L- amino 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 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 Major functional molecules Practical use

3. Protein-protein interaction network

5. Basic components

6. Peptide bond Planarity results from the delocalization of the lone pair of electrons of the nitrogen onto the carbonyl oxygen Torsion or dihedral angles 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

7. Protein structure Primary structure: amino acid sequence Secondary structure: Polypeptides are organized into hydrogen- bonded 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

9. 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).

10. PDB database Experimental Method ProteinsNucleic acids Protein/ Nucleic Acid complexes OtherTotal X-ray9242716454600498676 NMR96861124227811045 EM584291910804 Hybrid7032176 Other1664613189 Total:1029332805502626110790 August 2015

11. Examples of protein structures from PDB

12. Protein Engineering 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 Method to develop more useful or valuable proteins

13. 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

14. Biomolecular Eng. Lab. Random approach - Screening from nature - Random mutations Structure-based rational approach -Structure-function relationship -Site-directed/saturation mutagenesis Evolutionary approach - Directed evolution Accumulation of beneficial mutations No structural data HTS system Construction of diverse library 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 Technology Development

15. 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

16. 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

17. Glycan Profile of Glycoprotein Human (in Golgi) Pichia pastoris (in Golgi) ASN GlcNAc Man Gal Man GlcNAc Gal NANA 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 ASN GlcNAc Man

18. Fucose, Fuc C 6 O 5 H 12 Galactose, Gal C 6 O 6 H 12 Mannose, Man C 6 O 6 H 12 Sialic Acid, NAcNeuA C 11 O 9 NH 19 N-Acetylgalactosamine, GalNAc C 8 O 6 NH 15 N-Acetylglucosamine, GlcNAc C 8 O 6 NH 15 Monosaccharide structures

19. 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.

20. 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 %

21. Glycosylation pattern of EPO

22. 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) History of the EPO development

23. 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 New version of EPO by protein engineering

24. 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

25. 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

26. 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-methylglutaryl- coenzymeA (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.

27. The largest selling drug in the world : $ 12.9 billion in 2006 Generic drug : Simvastatin by Merck Lipitor

28. 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.

29. 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

30. 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. Halohydrin and Epoxide General structure of a halohydrin, where X = I, Br, F, or Cl An epoxide is a cyclic ether with three ring atoms Epoxide

31. Reaction scheme catalyzed by Halohydrin dehalogenase HCN

32. Tetramer Monomer

33. 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)

35. 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

36. Increase in the volumetric productivity : ~ 4,000 fold Development of a more economic process

37. 1 2 3 3 2 1 CDR FR VLVH Structural and functional features of Immunoglobulin Ab

38. 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

39. Engineering of Ab for humanization

40. Better tissue penetration, fast clearance, no effector function Local delivery Production in bacterial system Antibody Fragments

41. 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

42. Brand nameActive ingredient Type ClassTreatment Company 2013 global sales (US$ billion) Patent expiry EU/US Humira adalimumabAntibodyTNF inhibitorArthritis Abbott/Eisai10.7Apr 2018/ Dec 2016 Remicade infliximabAntibodyTNF inhibitorArthritis Merck/Mitsubishi8.9Aug 2014/ Sep 2018 Rituxan rituximabAntibodyAnti-CD20Arthritis, NHL Roche/Biogen8.6Nov 2013/ Dec 2018 Enbrel etanerceptAntibodyTNF inhibitorArthritis Amgen/Pfizer8.3Feb 2015/ Nov 2028 Lantus insulin glargineProteinInsulin receptorDiabetes Sanofi7.82014/2014 Avastin bevacizumabAntibodyAnti-angiogenesis Cancer Roche7.0Jan 2022/ Jul 2019 Herceptin trastuzumabAntibodyAnti-HER2Breast cancer Roche6.8Jul 2014/ Jun 2019 Neulasta pegfilgrastimProteinG-CSFNeutropenia Amgen4.4Aug 2017/ Oct 2015 Top 8 blockbuster biologicals (2013)

43. Fluorescent proteins

44. 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.

45. 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.

46. 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)

47. 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 )

48. Chromophore formation in GFP

49. 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

50. wtGFP : Ser(65)-Tyr(66)-Gly(67) Diverse Fluorescent Proteins by Protein Engineering

51. The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins. Fluorescence emission by diverse fluorescent Proteins

53. 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. Absorption and emission spectra