Bi1_2009_Lecture23_full - Putting biology to work for you...

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Unformatted text preview: Putting biology to work for you: In vitro (directed) evolution and other techniques Phage display • Make a combinatorial library of genes of interest • Put genes into a vector so that each gene product is expressed on the surface of a bacteriophage – Function encoded by each gene is on surface of phage – Gene is inside phage • Take collection of phage and select those with desired properties (e.g., binding to something) • Similar methods: yeast display, bacterial display, ribosome display See animation of phage display here: In vitro selection to produce human monoclonal antibodies or increase affinity of existing monoclonal antibody Figure A-15 Generate library of heavy and light chain variable regions using spleen DNA. Or introduce random mutations into variable regions genes of a specific antibody. Clone into a phage so Multiply phage that each phage display library in expresses one bacteria, bind phage VH-VL surface fusion to surface coated protein. with antigen. Wash away unbound phage. Repeat procedure (multiply recovered phage, bind to antigen, wash away unbound phage) for several cycles. Recover specific high-affinity antigen binding VH-VL regions. Phage Display schematic 1 3 2 4 5 6 7 8 Phage display to select for sequence-specific DNA binding proteins • Zinc finger proteins are modular. • Each finger contains two anti-parallel -strands, an -helix, and a Zn atom. • The -helix from each finger inserts into the major groove of DNA. A General Strategy for Selecting High Affinity Zinc Finger Proteins for Diverse DNA Target Sites Greisman et al., 1997, Science 275: 657 First use 3finger/DNA crystal structure to determine important protein/DNA contacts A General Strategy for Selecting High Affinity Zinc Finger Proteins for Diverse DNA Target Sites Greisman et al., 1997, Science 275: 657 Originally bound to this sequence RNA aptamers -- antibody-like properties Aptamers have been made against small molecules, peptides, proteins, organelles, viruses, cells Vitamin b12binding RNA aptamer Aptamer database: Let the immune system make enzymes for you Catalyst must bind more tightly to transition state than to products or reactants. Catalytic Antibodies • Raise antibodies against a transition state analog • Screen hybridomas for antibodies that catalyze desired reaction Wedemayer et al. (1997) Science 276, 1665-1669. Catalytic antibody that hydrolyzes cocaine Zhu et al., 2006, Structure 14: 205-216 (Nonpsychoactive products) Compound that was injected to raise antibodies Transition state analog that was crystallized with Fab / barrel enzymes evolved from a common ancestor 10% of enzymes are / barrels But this takes a long time… Directed evolution In vitro evolution of enzymes • Enzymes evolved for functions inside a living organism, not for biotechnology – Might need long-term stability – Might need activity in non-aqueous solutions • Produce new enzymes using recombinant DNA technology, but don’t know how by rational design • Use directed evolution Directed evolution experiment From Frances Arnold’s website: Some considerations… • Don’t start with random sequences because there are too many (20N) • Instead start with lightly mutagenized gene (e.g., error-prone PCR) or high level of random mutations to small part of gene Most mutations are destabilizing, so simply increasing protein stability can increase mutational robustness Marginally stable parent protein stable Stabilized parent protein unstable These previously unacceptable mutations are now acceptable. (critical threshold stability) Gf Gf “Protein stability promotes evolvability.” JD Bloom, ST Labthavikul, CR Otey, and FH Arnold. Proc Natl Acad Sci, 103:5869-5874 (2006). Evaluating stability Two state unfolding transition N <--> D Monitor property of folded protein as function of increasing temperature Transition midpoint (Tm) Shows that class I MHC molecules require bound peptide for thermal stability “Family” shuffling experiment From Frances Arnold’s website: From Frances Arnold’s website: Protein Library Design multiple sequences 1 structure Sequence Space Structure Space Mayo lab, Caltech Protein Fold Prediction versus Protein Design 1 sequence 1 structure Sequence Space Structure Space Mayo lab, Caltech Computational Protein Design: Rationale by the Numbers Combinatorial Explosion 1 protein p residues 20 amino acid types 20 p sequences Residues 18 37 42 59 Sequences 1023 1048 1054 1077 Mass Baseball Earth Sun Universe Mayo lab, Caltech Methods Atom-Based Forcefields Rotamer Libraries Protein Backbones Combinatorial Optimization Algorithms Negative Design Computational Protein Design Optimization of Rotamers by Iterative Techniques (ORBIT) Apply to protein fold stabilization, enzyme design Applications Mayo lab, Caltech De Novo Protein Design: Fully automated sequence selection Dahiyat & Mayo, 1997, Science 278: 82-87 Designed protein Zif268 Zn finger Target fold: Zif268 (a zinc finger) Comparison of original and designed protein structures Stability Based Design: Protein G • Bacterial protein involved in host immune system evasion 56 amino acid domain Objective was to stabilize protein while preserving structure and function Design focused on 26 core and boundary positions Combinatorial complexity, 106 amino acid sequences Mayo lab, Caltech • • • • Kemp Elimination: a model system for enzyme design O O O H O HO N O H O2N N O O2N N O C O2N O- 5-nitrobenzsoxazole catalytic antibody: kcat/kuncat = 106 ab initio t.s. model active site template General Base Asp/Glu O H O O N * * Phe/Trp N O O H-bond donor Hie/Hid/Gln/Asn HN N H O H-bond donor Ser/Thr/Tyr Thorn et al., Nature 1995 Debler et al., PNAS 2005 Hu et al., JACS 2004 Mayo lab, Caltech ...
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This note was uploaded on 09/25/2010 for the course BIO 1 taught by Professor Bakorman during the Spring '09 term at Caltech.

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