Introduction

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 cog-nate proteins / ligands

- Cell signaling processes • Enzymes catalyze the reactions with high specificity and catalytic effi-

ciency 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

Protein-protein interaction network

Basic components

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

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

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

ExperimentalMethod Proteins Nucleic acids

Protein/Nucleic Acidcomplexes

Other Total

X-ray 92427 1645 4600 4 98676NMR 9686 1124 227 8 11045EM 584 29 191 0 804Hybrid 70 3 2 1 76Other 166 4 6 13 189

Total: 102933 2805 5026 26 110790

August 2015

Examples of protein structures from PDB

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

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 pro-teins, Protein binders

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

New version of therapeutic proteins by Protein Eng

• EPO (Erythropoietin) with a longer plasma half-life by incorporation of addi-tional 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

Human(in Golgi)

Pichia pastoris(in Golgi)

ASN GlcNAc GlcNAc Man

Gal

ManGlcNAc

GlcNAc

Gal NANA

NANAMan

• Glycan profile: very complex and varies broadly, depending on cell types and pro-duction 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 path-way, and property of glycoproteins are a key to Glycobiology

ASN GlcNAc GlcNAc Man

Man

Man

Man

Man

Man

Man

Man Man

Man Man

Man Man

Man

Fucose, FucC6O5H12

Galactose, GalC6O6H12

Mannose, ManC6O6H12

Sialic Acid,NAcNeuAC11O9NH19

N-Acetylgalactosamine,GalNAc

C8O6NH15

N-Acetylglucosamine,GlcNAc

C8O6NH15

Monosaccharide structures

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 ac-

tivates a JAK2 signaling cascade• Clinically used in treating anemia resulting from chronic kidney disease, in-

flammatory bowel disease (Crohn's disease and ulcer colitis), and myelodys-plasia 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

• 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

• 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

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 consen-sus 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 re-ductase)

- HMG-CoA reductase catalyzes the reduction of 3-hydroxy-3-methylglutaryl-coen-zymeA (HMG-CoA) to mevalonate, which is the rate-limiting step in hepatic choles-terol biosynthesis.

• Inhibition of the enzyme decreases de novo cholesterol synthesis, increasing expres-sion 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 pa-tients.

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 con-

trolled. The hydroxyl in HN is defined the second stereo-center in atorovas-tatin, and high chemical purity is essential for downstream chemistry

Essential for more economic process

• A type of organic compound or functional group in which one carbon atom has a halogen substituent, and an adjacent carbon atom has a hy-droxyl 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

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

1 2

33

2

1CDR

FR

VL VH

Structural and functional features of Immunoglobulin Ab

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

• Better tissue penetration, fast clearance, no effector function• Local delivery• Production in bacterial system

Antibody Fragments

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

Brand name Active ingredient

Type Class Treatment Company 2013 global sales(US$ billion)

Patent expiryEU/US

Humira adalimumab Antibody TNF inhibitor Arthritis Abbott/Eisai 10.7 Apr 2018/ Dec 2016

Remicade infliximab Antibody TNF inhibitor Arthritis Merck/Mitsubishi 8.9 Aug 2014/ Sep 2018

Rituxan 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

Top 8 blockbuster biologicals (2013)

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 moni-

tor 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

wtGFP : Ser(65)-Tyr(66)-Gly(67)

Diverse Fluorescent Proteins by Protein Engineering

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

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 fluores-cent protein (DS Red2, AS Red2, HC Red). From Clontech.

Absorption and emission spectra