Protein Engineering Protocols - Mycobacteriology research center

Index 307parameters in creation and screening,biased libraries, 130, 131experimental biases, 133library representation, 132, 133size,design, 129, 130limits, 131, 132phage display, see Phage displayprobability calculations,library representation,equiprobable outcomes, 139–144, 149, 150experimental bias testing withχ 2 test, 148, 149, 152overview, 138, 139unequal probability outcomes,144–147, 151, 152software, 133Compartmentalized self-replication (CSR),principles, 239–242Taq polymerase engineering,bacterial expression, 243materials, 242, 243polymerase chain reaction,244, 246pull through, 245, 246quick screening of variants,245, 246setup, 243, 244, 246work-up of reaction,polyethylene glycol extraction,244–246quick work-up, 245Computational protein design,coiled coils, 59–61, 64combinatorial libraries, seeCombinatorial librariesdirected protein design, 4examples, 5, 6limitations, 6probabilistic protein design, 6, 7profile building, 8, 9sequence alignment, 8statistical theory of sequenceensembles, backboneflexibility, 9, 17, 18conformational entropy, 9, 10constrained optimization ofentropy, 10, 18energy functions, 10, 11reference energy, 12, 13rotamer and identity probabilities,13, 15solvation and hydrophobicenergy, 11, 12CSR, see Compartmentalizedself-replicationDDegenerate oligonucleotide geneshuffling (DOGS),chimeric gene amplification, 197, 199cloning of shuffled products, 199materials, 192, 193plate assays with Congo Red, 199,200, 202polymerase chain reaction,degenerate primers,design, 193–195, 197, 202features, 195–197, 201, 202gene-specific nested end primerdesign, 195overlap extension of genesegments, 197, 202principles, 192xylanase assay, 200, 201DHFR PCA, see Dihydrofolatereductase protein-fragmentcomplementation assayDihydrofolate reductase proteinfragmentcomplementationassay (DHFR PCA),principles, 251Ras-binding domain of Raf librarypreparation and selection,