1 Module 2 overview lecture lab 1 Introduction to the module 1 Start up protein eng 2 Rational protein design 2 Site directed mutagenesis 3 Fluorescence and sensors 3 DNA ampli cation 4 Protein expression 4 Prepare expression system SPRING BREAK 5 Review gene analysis 5 Gene analysis induction 6 Puri cation and protein analysis 6 Characterize expression 7 Binding af nity measurements 7 Assay protein behavior 8 High throughput engineering 8 Data analysis 2 Lecture 8 High throughput engineering I General requirements for HT engineering A High throughput vs rational design B Generating libraries II Selection techniques A Phage display and related techniques B Selection for properties other than af nity 3 4 Rational protein design Knowldege based deterministic engineering of proteins with novel characteristics Irrational high throughput protein engineering Selection for desired properties from libraries of random variants 5 Methods for generating mutant protein libraries site directed mutagenesis with degenerate primers error prone PCR gene shuf ing Degenerate primers gat aag gac ggc gat gcc acg att acc acc D K D G D G T I T T ga c g D E xcc S P T A xxx X not all combinations of AA s possible at each position number of combinations expands exponentially degenerate primers synthesized by split pool method standard primer design criteria must be considered PCR polymerase and conditions may be chosen to promote mutations 6 error rate mutation load template doublings normal PCR error prone PCR some mutations are more likely than others Tables from Biles B D and B A Connolly Nucleic Acids Research 32 no 22 2004 e176 Oxford University Press All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse 7 Gene shuf ing techniques mimic diversity due to meiotic recombination fragments of homologous genes combined using sexual PCR diversity may arise from error prone PCR or multiple genes Reprinted by permission from Macmillan Publishers Ltd Nature Reviews Molecular Cell Biology Source Brannigan J A and A J Wilkinson Protein Engineering 20 Years on Nat Rev Mol Cell Biol 3 2002 964 70 2002 Brannigan Wilkinson 2002 Nat Rev Mol Cell Biol 3 964 70 How are libraries of mutant proteins screened All major methods include a strategy to keep DNA sequence info associated with the proteins that are being screened Phage display is a versatile high throughput method to do this protein displayed on the coat of a bacteriophage by fusing to a natural phage coat protein M13 phage contains DNA Image from Branden C and J Tooze Introduction to Protein Structure 2nd ed New York NY Garland Science 1999 Garland Science All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse 8 9 Application phage displayed peptides that bind to GaAs selected sequences phages patterned on target substrate Whaley et al 2000 Nature 405 665 8 Reprinted by permission from Macmillan Publishers Ltd Nature Source Whaley S R et al Selection of Peptides with Semiconductor Binding Specificity for Directed Nanocrystal Assembly Nature 405 2000 665 668 2000 10 Yeast display similar to phage display but with proteins fused to a Saccharomyces cell wall protein DNA in yeast What would you expect advantages to be compared with phage display In this example a population of scFvs was screened for binding to an antigen left selection criterion for FACS assay right comparison of wt blue and selected red scFv binding Chao et al 2006 Nat Protoc 1 755 68 Reprinted by permission from Macmillan Publishers Ltd Nature Protocols Source Chao G et al Isolating and Engineering Human Antibodies Using Yeast Surface Display Nature Protocols 1 2006 755 768 2006 11 Ribosome display mRNA and synthesized proteins held together noncovalently on a ribosome What are advantages of this technique over phage yeast display methods screening not in the presence of large particles incorporation of unnatural amino acids American Chemical Society All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse Left Hanes Pl ckthun 1997 Proc Natl Acad Sci USA 94 4937 42 Right Josephson et al 2005 J Am Chem Soc 127 11727 35 Courtesy of National Academy of Sciences U S A Used with permission Source Hanes J and A Pl ckthun In Vitro Selection and Evolution of Functional Proteins by Using Ribosome Display PNAS 94 no 10 1997 4937 4942 Copyright 1997 National Academy of Sciences U S A What about properties other than af nity A simple example screen for dsRed variants with different excitation and emission wavelengths how could this be done Shaner et al 2004 Nat Biotechnol 22 1567 72 Reprinted by permission from Macmillan Publishers Ltd Nature Biotechnology Source Shaner N C et al Improved Monomeric Red Orange and Yellow Fluorescent Proteins Derived from Discosoma sp red Fluorescent Protein Nature Biotechnology 22 2004 1567 1572 2004 12 Directed evolution of enzymatic activity screen is a uorescence assay 13 Joo et al 1999 Nature 399 670 3 Reprinted by permission from Macmillan Publishers Ltd Nature Source Joo H et al Laboratory Evolution of Peroxide Mediated Cytochrome P450 Hydroxylation Nature 399 1999 670 673 1999 Which type of screening method to use screen method throughput other notes SELEX 1015 selection of DNA RNA ribosome display 1015 in phage display 1011 best for small proteins peptides yeast display 108 compatible w eukar proteins plate assays 105 vitro protein synthesis versatile but more complex number of variants in a protein library x residues 20x possible variants 12 residues 4 x 1015 variants lesson impossible to cover sequence space except with short sequences or few positions and only the most high throughput techniques 14 MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing 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