3. Protein Engineering BÍ7430 Molecular Biotechnology Outline □ Limitations of proteins in biotech processes □ Definition and aim of protein engineering □ Targeted properties of proteins □ Approaches in protein engineering ■ Directed evolution ■ Rational design Proteins in biotechnology □ main problem - identify or design optimal protein for specific process historically - adapt process future - adapt protein Proteins in biotechnology □ classical screening screening culture collections polluted and extreme environment □ environmental gene libraries metagenomic DNA □ data-base mining genome sequencing projects massive sequencing (Sargasso sea project) numerous uncharacterised enzymes □ protein engineering Protein engineering □ 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, Kt) cofactor selectivity protein-protein or protein-DNA interactions 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) only improvements (molecular evolution) does not take millions of years, but happens rapidly Principal of directed evolution evolution in test tube comprises essentially 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 expession system screening or selection system $ Improved protein I Random mutagenesis ooocooo i • i:..n:.J vi:.-,-. i 3 Transformation 4, Protein expression 5. uatippliriJ O SLrwiriirig una sanction - activity ', Biochemical testing $$$$ Methods to create mutant libraries Jj feci □ technology to generate large diversity NON-RECOMBINING (one parent gene -> variants with point mutations) t RECOMBINING (several parental homologous genes -> chimeras) V Non-recombining mutagenesis □ UV irradiation or chemical mutagens (traditional) □ mutator strains - lacks DNA repair mechanism mutations during replication (e.g., Epicurian coli XLl-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 o 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 n I* fragmentation step * random reassembly of segments -.-E3*^" □ StEP - staggered extension process simpler then shuffling Jj-B random reannealing combined with limited primer extension ~-—' ~_ □ other methods n shuffling of genes with lower homology down to 70% (e.g., RACHITT, ITCHY, SCRATCHY) l.-jtj=,h L 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) 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) 104 libraries ■ volume 10 - 100 uL □ microfluidic systems water in oil emulsions (up to 10 kHz) FACS sorting (10a events/hour) 10 libraries volume 1 - 10 pL ~1 9 f- Direct selection from mutant library □ not generally applicable (mutant libraries >10 variants) □ link between genotype and phenotype □ display technologies -^—^S^ phage display /* ribosome display ^"^"'^t □ life-or-death assay auxotrophic strain ■3' L Example of Directed evolution □ directed evolution of enantioselectivity lipase from P. aeruginosa (E-value improved from 1.1 into 51) spectrophotometry screening of (R)- and (5)-nitrophenyl esters the best mutant contains six amino acid substitutions 40 000 variants screened L Approaches in protein engineering I ^ \3 -- RATIONAL DESIGN 1. Computer aided des gn :-V'b* , _ 2, Site rlirsrwl mutagenes! a i. Translonnaticn A. Protein exp riss i ?r 5, Pr.H.Mi |.unhirv.r DIRECTED EVOLUTION $ Improved protein 7, Bloc Fen let I lesllnc 2. Random mutagenesis OOOCOÜO 3, Transformation 4. Protein *xp rare ion =. Screening and (election lilirhd nJiitiufiin Rational design □ emerged around 1980s as the original protein engineering approach □ combining theory (knowledge based) and experiment □ protein engineering cycle: ..structure-theory-design-mutation-purification-analysis" □ difficulty in prediction of mutation effects on protein property Principal of rational design 1. Co ri .l top .i i-.II d*sign 2. Site-di reeled muta genes is 3. ransttrmation A. Pi-e.tÉiii **pre-M in Ě P retain purification -°"BM Improved .V, protein ■vi-ířni-titi** T. Biochamicel mating 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, IMMR) understanding between structure and function 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 differences 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, IMMR, homology models) molecular modelling Construction -r—7TH -—_A^r^'-/s □ site-directed mutagenesis introducing point mutations QuickChange Kit (one day method) ").,J"""8..p^..... □ multi site-directed mutagenesis 66 □ gene synthesis 6 commercial service codone optimisation .....a.siinsi::':.....i:s Example of rational design □ rational design of protein stability stability to high temperature, extreme pH, oxygen stress, proteases etc. stabilizing mutations increase strength of weak interactions 0 salt bridges and H-bonds (E/js/nk eta/., B/ochem. J. 285: 625-628, 1992) 0 S-S bonds (Matsumura eta/., Nature 342: 291-293, 1989) 0 addition of prolines (Watanabe eta/., Eur. 3. B/ochem. 226: 277-283, 1994) 0 less glycines (Margar/t eta/., Protein Eng. 5: 543-550, 1992) 0 oligomerisstion (Da/hus eta/., 3. Mo/. Bio/. 318 : 707-721, 2002) Example of rational design □ rational design of protein stability engineering protein to resist boiling {Burg et a/., pnas 95: 2056-2060, 1998) 0 reduced rotational freedom - Thr56Als, Gly58Als, Ser65Pro and Als96Pro 0 introduction of disulfide bridge - GlySCys + Asn60Cys 0 improved internal hydrogen bond - Als4Thr 0 filling cavity - Tyr63Phe