Bi7430 Molecular Biotechnology
4. Protein Engineering
Outline
 Limitations of proteins in biotechnology processes
 Definition and aim of protein engineering
 Targeted properties of proteins
 Basic approaches in protein engineering
 DIRECTED EVOLUTION
 RATIONAL DESIGN
 SEMI-RATIONAL DESIGN
 Examples
Proteins in biotechnology
Available protein
Available protein
Suitable/adopted protein
 availability of optimal protein for specific process
 traditional biotechnology - adapt process
 modern biotechnology - adapt protein
H O W TO O BTA I N O P T I M A L P R OT E I N ?
Proteins in biotechnology
 classical screening
 screening culture collections
 polluted and extreme environment
 environmental gene libraries
 metagenomic DNA
 data-base mining
 gene databases
 genome sequencing projects
 numerous uncharacterised enzymes/proteins
 PROTEIN ENGINEERING
I F S U I TA B L E P R OT E I N D O E S N OT E X I S T I N N AT U R E ?
Proteins in biotechnology
 the process of constructing novel protein molecules
by design first principles or altering existing structure
 use of genetic manipulations to alter the coding sequence of a
gene and thus modify the properties of the protein
 AIMS AND APPLICATIONS
 technological - optimisation of the protein to be suitable in
particular technology purpose
 scientific - desire to understand what elements of proteins
contribute to folding, stability and function
Targeted properties of proteins
 structural properties of proteins
 stability (temperature, solvents)
 tolerance to pH, salt
 resistance to oxidative stress
 functional properties of proteins
 reaction type
 substrate specificity and selectivity
 kinetic properties (e.g., Km, kcat, Ki)
 cofactor selectivity
 protein-protein or protein-DNA
interactions
Strategies in protein engineering
Improved
protein
Directed evolution
 directed evolution techniques emerged during mid -1990s
 inspired by natural evolution
 this form of "evolution" does not match what Darwin had envisioned
 requires outside intelligence, not blind chance
 does not create brand new species, macroevolution,
but only improvements of molecules, molecular evolution
 does not take millions of years, but happens rapidly
Improved
protein
Directed evolution
 evolution in test tube comprises two steps
 random mutagenesis
mutant library building
 screening and selection
identification of desired biocatalyst
 prerequisites for directed evolution
 gene encoding protein of interest
 method to create mutant library
 suitable expression system
 screening or selection system
Methods to create mutant libraries
 technology to generate large diversity
 NON-RECOMBINING
one parent gene -> variants with point mutations
 RECOMBINING
several parental homologous genes -> chimeras
Non-recombining mutagenesis
 UV irradiation or chemical mutagens (traditional)
 mutator strains - lacks DNA repair mechanism
mutations during replication (e.g., Epicurian coli XL1-Red)
 error-prone polymerase chain reaction (ep-PCR)
 gene amplified in imperfect copying process
(e.g., unbalanced deoxyribonucleotides concentrations,
high Mg2+ concentration, Mn2+, low annealing temperatures)
 1 to 20 mutation per 1000 base pairs
 saturation mutagenesis
 randomization of single or multiple codons
 other methods
 gene site saturation mutagenesis
 cassette mutagenesis (region mutagenesis)
Recombining mutagenesis
 also refered to as „sexual mutagenesis“
 DNA shuffling
 fragmentation step
 random reassembly of segments
 StEP - staggered extension process
 simpler then shuffling
 random reannealing combined with
limited primer extension
 other methods
shuffling of genes with lower homology down to 70%
(e.g., RACHITT, ITCHY, SCRATCHY)
Screening and selection
 most critical step of direct evolution
 isolation of positive mutants hiding in library
 HIGH THROUGHPUT SCREENING
individual assays of variants one by one
 DIRECT SELECTION
display techniques (link between genotype and phenotype)
(Utra)High throughput screening
 common methods not applicable
 agar plate (pre)screening
 microtiter plates screening
 96-, 384- or 1536-well formate
 robot assistance
(colony picker, liquid handler)
 10
4
libraries
 volume 10 – 100 uL
 microfluidic systems
 water in oil emulsions ( u p t o 1 0 k H z )
 FACS sorting (1 0 8 e v e n t s / h o u r )
 10
9
libraries
 volume 1 – 10 pL
Direct selection
 not generally applicable (mutant libraries >10
6
variants)
 link between genotype and phenotype
 display technologies
 ribosome display
 phage display
 life-or-death assay
 auxotrophic strain
 toxicity based selection
Example of Directed evolution
 directed evolution of enantioselectivity
 lipase from P. aeruginosa (E-value improved from 1.1 into 51)
 spectrophotometric screening of (R)- and (S)-nitrophenyl esters
 40 000 variants screened
 the best mutant contains six amino acid substitutions
Strategies in protein engineering
Improved
protein
Rational design
 emerged around 1980s as the original protein engineering approach
 knowledge based - combining theory and experiment
 protein engineering cycle:
„structure-theory-design-mutation-purification-analysis“
 difficulty in prediction of mutation effects on protein property
 de novo design
Principal of rational design
Improved
protein
 rational design comprises:
 design - understanding of protein
functionality
 experiment - construction and testing
of mutants
 prerequisites for rational design:
 gene encoding protein of interest
 3D structure (e.g., X-ray, NMR)
 structure-function relationship
 computational methods and capacity
 (multi)side directed mutagenesis
techniques
 efficient expression system
 biochemical tests
Design
 HOMOLOGY APPROACH
 homologous wild-type sequences are collected and compared
 identifying amino acid residues responsible for difference s
 reconstruction - transfer differences from one enzyme to another
 new design - combination of possitive mutation from all parental
proteins in one construct, new protein better than all parental
Design
 STRUCTURE-BASED APPROACH
 prediction of enzyme function from structure alone is challenging
 protein structure (X-ray crystallography, NMR, homology models)
 molecular modelling
o molecular docking
o molecular dynamics
o quantum mechanics/molecular mechanics (QM/MM)
Construction
 site-directed mutagenesis
 introducing point mutations
 multi site-directed mutagenesis
 gene synthesis
 commercial service
 codone optimisation
Example of rational design
 rational design of protein stability
 stability to high temperature, extreme pH, proteases etc.
 stabilizing mutations increase strength of weak interactions
o salt bridges and H-bonds
Eijsink et al., Biochem. J. 285: 625-628, 1992
o S-S bonds
Matsumura et al., Nature 342: 291-293, 1989
o addition of prolines
Watanabe et al., Eur. J. Biochem. 226: 277-283, 1994
o less glycines
Margarit et al., Protein Eng. 5: 543 -550, 1992
o oligomerisation
Dalhus et al., J. Mol. Biol. 318: 707 -721, 2002
Example of rational design
 engineering protein to resist boiling
 reduced rotational freedom
Thr56Ala, Gly58Ala, Ser65Pro and Ala96Pro
 introduction of disulfide bridge
Gly8Cys + Asn60Cys
 improved internal hydrogen bond
Ala4Thr
 filling cavity
Tyr63Phe
Burg, B., et al., 1998. PNAS 95: 2056-2060
Half-lifes
(min.)
80°C 100°C
wild type 17.5 >0.5
8-fold
mutant
stable 170
Strategies in protein engineering
Strategies in protein engineering
Strategies in protein engineering
S E M I R AT I O N A L
D E S I G N
Example of rational design
 c o n v e rs i o n o f 1 , 2 , 3 - t r i c h l o ro p ro p a n e
b y D h a A f ro m R h o d o c o c c u s e r y t h r o p o l i s Y 2
 D I R E C T E D E VO LU T I O N - i m p o r t a n c e o f a c c e s s p a t h w ay s
C176
Y176
F176
Bosma, T., et al. 2002: AEM 68: 3582-87 Gray, K.A., et al. 2003: Adv. Appl. Microbiol. 52: 1-27
Example of rational design
Pavlova, M., Klvana, M., Prokop, Z., et al. 2009: Nature Chem. Biol. 5: 727-733
 c o n v e rs i o n o f 1 , 2 , 3 - t r i c h l o ro p ro p a n e
b y D h a A f ro m R h o d o c o c c u s e r y t h r o p o l i s Y 2
 D I R E C T E D E VO LU T I O N - i m p o r t a n c e o f a c c e s s p a t h w ay s
 S E M I - R AT I O N A L D E S I G N - h o t s p o t s i n a c c e s s t u n e l s
 l i b ra r y o f 5 , 3 0 0 c l o n e s s c re e n e d
Example of rational design
Pavlova, M., Klvana, M., Prokop, Z., et al. 2009: Nature Chem. Biol. 5: 727-733