Chem170Lecture10

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1、Chemistry170:ChemicalBiologyLecture10HarvardUniversityFall2003ProfessorDavidLiuMolecularEvolution(Part2:CaseStudies)•Enhancingoralteringnaturalfunction(continued)!–Greenfluorescentprotein(GFP)–Aminoacyl-tRNAsynthetases–Tetracyclinerepressor(TetR)•Evolvingnewfunction–Remodelingprote

2、ins:hGHandproteinA–Erythropoietinmimetics–Protease-resistantpeptideligandsABrighterGreenFluorescentProtein(Crameri,Stemmer,1996)•Greenfluorescentprotein(GFP)isextremelyusefulasaproteintagandasareporterofgeneregulation•Goal:createamutantGFPthatisbrighterinvivo–ThequantumyieldofwtGFP

3、isalready0.7-0.8–ExpressionlevelsinE.coliarealready75%oftotalprotein–Therefore,notclearhowtosolvethisproblemrationally•Firststep:optimizecodonusage;thisyieldsa3-foldimprovement•Diversity:DNAshuffling•Tagging:cells(E.coli)•Picking:invivoscreen(platecoloniesexpressingmutantGFPs,shine

4、aUVlampovertheplates,andpickthebrightestcolonies)Results:ABrighterGFP•40brightestcoloniespickedfromlibrariesof10,000transformants•AftertworoundsofDNAshufflingandscreening:23-foldmorefluorescentcellscomparedwiththestartingconstruct•Threerounds:45-foldmorefluorescent•Excitationandemi

5、ssionpeaksunshiftednoGFPwtGFPoptim.wtround2round3GFPGFPGFPBasisforEnhancedGFPFluorescence•Wild-typeGFPisnaturallyboundtoaprotein(aequorin),likelyinvolvingafewermutations!hydrophobicinterface•Intheabsenceofaequorin,GFPmayaggregateatthisinterface,leadingtoinsolubleinclusionbodiesandl

6、owercellfluorescence•Consistentwiththis,wtGFPisexpressedasmostlyinsoluble•Round3evolvedGFP:containsthreemutations(Phe100Ser,Met154Thr,Val164Ala)whichintroducelesshydrophobicresidues•Round3GFPismostlysoluble,unlikewtGFP,partiallyexplainingpropertiesUnnaturalAminoAcidMutagenesisinLiv

7、ingCells(Schultzetal.,1999-2003)IsitpossibletoexpandthegeneticcodeofE.coli?Requirements:•An“orthogonal”tRNAthatisnotasubstrateforcellularaaRSs•An“orthogonal”aaRSenzymethatchargestheorthogonaltRNAwiththeunnaturalaminoacidbutnoneofthenaturalaminoacids•Anunnaturalaminoacidthatistakenu

8、pbythecell•StartwithatRNA/