, LOSCHMIDT , LABORATORIES Protein Engineering BÍ7430 Molecular Biotechnology 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 □ key problem -availability of optimal protein for specific process □ traditional biotechnology - adapt process □ modern biotechnology - adapt protein Proteins in biotechnology □ classical screening ■ screening culture collections ■ polluted and extreme environment □ environmental gene libraries ■ metagenomic DNA □ data-base mining ■ gene databases ■ (meta)genome sequencing projects ■ numerous uncharacterised proteins 250 -Gene Bank -Swissprot 1990 1997 2004 IF SUITABLE PROTEIN DOES NOT EXIST IN NATURE? □ PROTEIN ENGINEERING Proteins in biotechnology □ the process of constructing novel protein molecules by design first principles or altering existing structure „de novo design" □ 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, /C,) ■ cofactor selectivity ■ protein-protein or protein-DNA interactions Reaction coordinate Strategies in protein engineering RATIONAL DESIGN 1. Computer aided design ■ \ 2. Site-directed mutagenesis Individual mutated gene 3. Transformation 4. Protein expression 5. Protein purification 6. noř applied DIRECTED EVOLUTION 1. not applied \ I m proved protein Constructed mutant enzyme 7. Biochemical testing 2. Random mutagenesis Library of mutated genes (>10,000 clones ) 3. Transformation 4. Protein expression 5. not applied 6. Screening and selection - stability - selectivity - affinity - activity Selected mutant enzymes 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 Directed evolution □ evolution in test tube comprises two steps ■ random mutagenesis building mutant library (diversity) ■ 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 I m proved protein 1. not applied 2. Random mutagenesis DC Library of mutated genes ( >10,000 clones ) 3. Transformation 4. Protein expression 5. not applied 6. Screening and selection -stability - selectivity - affinity - activity 7. Biochemical testing Selected mutant enzymes 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 I I I Iii 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 ■ randomization of single or multiple codons ■ gene site saturation mutagenesis □ other methods ■ insertion/deletions (InDel) ...... 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) A- 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) library of genes in test tube bacterial colonies on agar plate ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo mutant enzymes in nutrient broth OOOOOOOOOOOOO OOOOOOOOOOOOO OOOOOOOOOOOOO OOOOOOOOOOOOO ooooooooooooo ooooooooooooo ooooooooooooo ooooooooooooo visualisation of possitive mutants (Utra)High throughput screenin □ common methods not applicable □ agar plate (pre)screening □ microtiter plates screening ■ 96-, 384- or 1536-well formate ■ robot assistance (colony picker, liquid handler) 10 libraries volume 10 - 100 uL □ microfluidic systems (Lesson 5) ■ water in oil emulsions (up to 10 kHz) ■ FACS sorting (108 events/hour) 10 libraries volume 1 - 10 pL I C3 Nozzli tip Laser Bc.ini ■ Pninl c', analysis Break off point - Deflection jj • ptatl. • 1 *0 . Direct selection □ not generally applicable (mutant libraries >10 variants) □ link between genotype and phenotype □ display technologies ■ ribosome display ■ phage display □ life-or-death assay ■ auxotrophic strain ■ toxicity based selection RT. PCR Dissociation of ribosomes Transcription Tailored folding conditions Example of Directed evolution □ directed evolution of enantioselectivity ■ lipase from P. aeruginosa (E-value improved from 1.1 into 51) ■ spectrophotometric screening of (/?)- and (S)-nitrophenyl esters ■ 40 000 variants screened ■ the best mutant contains six amino acid substitutions Reetz, M., et al. 2001. Angew. Chem. Int. Ed. 40: 3589-91 Rational design □ emerged around 1980s as the original protein engineering approach □ knowledge based - combining theory and experiment □ protein engineering cycle: „ st ructu re -theory -design-mutation-purification-ana lysis" □ difficulty in prediction of mutation effects on protein property □ de novo design most challenging Principal of rational design I. Computer aided design "ft___ f 2. Site-directed mutagenesis Individual mutated gene 3. Transformation 4. Protein expression 5. Protein purification 6. not applied I mproved protein Constructed mutant enzyme 7. 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J. 285: 625-628, 1992 o S-S bonds Matsumura et al., Nature 342: 291-293, 1989 o addition of prolines f~ Watanabe et al., Eur. J. Biochem. 226: 277-283, 1994 ^N H 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 Ser65Pro, Ala96Pro ■ introduction of disulfide bridge Gly8Cys + Asn60Cys ■ improved internal hydrogen bond Ala4Thr ■ filling cavity Tyr63Phe Half-lifes (min.) 80°C 100°C wild type 17.5 >0.5 mutant stable 170 Burg, B., et al., 1998. PNAS 95: 2056-2060 Strategies in protein engineering RATIONAL DESIGN 1. Computer aided design 2. Site-directed mutagenesis Individual mutated gene 3. Transformation 4. Protein expression 5. Protein purification 6. not applied Constructed mutant enzyme IMPROVED ENZYME 7. Biochemical testing DIRECTED EVOLUTION SEM I RATIONAL DESIGN 2. Random mutagenesis Library of mutated genes ( > 10,000 clones ) 3. Transformation 4. Protein expression 5. not applied 6. Screening and selection - stability - selectivity - affinity - activity ^^^^ ^^^^ Selected mutant enzymes Example of semi-rational design □ conversion of 1, 2,3-t r i c h I o r o p ro p a n e by DhaA from Rhodococcus erythropoIis Y2 Example of semi-rational design □ conversion of 1, 2,3-t r i c h I o r o p ro p a n e by DhaA from Rhodococcus erythropoIis Y2 □ DIRECTED EVOLUTION - importance of access pathways C176 Bosma, J., et al. 2002: AEM 68: 3582-87 Gray, K.A., et al. 2003: Adv. Appl. Microbiol. 52: 1-27 Example of semi-rational design □ conversion of 1, 2,3-t r i c h I o r o p ro p a n e by DhaA from Rhodococcus erythropoIis Y2 □ DIRECTED EVOLUTION - importance of access pathways □ SEMI-RATIONAL DESIGN - hot spots in access tunels □ library of 5,300 clones screened Pavlova, M., Klvana, M., Prokop, Z., et al. 2009: Nature Chem. Biol. 5: 727-733 Example of semi-rational design 1.2 n Reaction coordinate Pavlova, M., Klvana, M., Prokop, Z., et al. 2009: Nature Chem. Biol. 5: 727-733 Example of semi-rational design STANDARD DESIGN random mutagenesis (2-3 positions) I ibrary of 1 04 clones volume: 100' \xL assays/day: 103 ADVANCED DESIGN random mutagenesis (5-7 positions) I ibrary of >1 06 clones volume: 10' pL assays/day: 107 Reading □ Lutz, S. 2010: Beyond directed evolution - semi-rational protein engineering and design. Curr Opin Biotechnol. 21(6): 734-743 □ Computational enzyme redesign and Computational de novo enzyme design (page 5-7) /t>t NIH Public Access Author Manuscript > > o Published in final edited form as: Curr Opin Biotechnol 2010 December ; 21(6): 734-743. doi:10.10l6/j.copbio.2010.08.011. Beyond directed evolution - semi-rational protein engineering and design Stefan Lutz Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA: 30322 Abstract Over the last two decades, directed evolution lias transformed the field of protein engineering. The advances in understanding protein structure and function, in no insignificant part a result of directed evolution studies, are increasingly empowering scientists and engineers to device more effective methods for manipulating and tailoring biocatalysts. Abandoning large combinatorial libraries, the