Transcript Protein Engineering

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.