Protein Engineering Protocols - Mycobacteriology research center

310 Indexmaterials, 169, 170mutation rate analysis, 180, 182NExTProg for fragmentationprediction,calibration and comparison withexperimental results, 185–187mathematics, 183, 188overview, 182, 183principles, 168uridine-exchange polymerase chainreaction, 171, 173, 187PPCA, see Protein-fragmentcomplementation assayPCR, see Polymerase chain reactionPeptide Velcro hypothesis, see Coiled coilPhage display,coiled coil selection, 59library screening, 250M13 coat proteins,P8 engineering for improveddisplay,dU-single-stranded DNAtemplate preparation, 210,211, 217electroporation and phagepropagation, 214, 215, 218heteroduplex closed circularDNA synthesis, 212–214, 218library design, 209, 210materials, 206–208overview, 206, 208selection and analysis ofmutants, 216, 217stop template phagemid, 210, 217types for fusion, 205, 208monobody library sorting,panning, 103, 104phage clone characterization, 104ribosome-inactivation displaysystem, see Ribosomeinactivationdisplay systemPolymerase chain reaction (PCR),compartmentalized self-replication,244, 246nucleotide exchange and excisiontechnology and uridineexchangepolymerase chainreaction, 171, 173, 187random mutagenesis, 285, 286, 300ribosome-inactivation displaysystem, 233Protein-fragment complementationassay (PCA),coiled coil selection, 58, 59dihydrofolate reductase, seeDihydrofolate reductaseprotein-fragmentcomplementation assayprinciples, 251steric requirements, 255, 257Protein thermostability,compensatory mutation by directedevolution, 277rational design, 276rationale for engineering, 275, 276terminal truncation–directedevolution–re-elongation forenzyme enhancement,DNA shuffling and randommutagenesis, 285, 286, 300enzyme assay,kinetic parameterdetermination, 294spectrophotometric assay,293, 301thermoactivity profiling, 294,296, 301full-length mutant construction,290, 291β-lactamase as model system,281, 282materials, 279–281plasmid design, 282–284principles, 275–277, 279