简介：Astructuralgene(750bp),whichcodesforatypeIribosomeinactivatingprotein,trichosanthin,hasbeendesignedaccordingtothecodonusageofhighlyexpressedgeneinE.coliandchemicallysynthesized.Inthesynthesizedgene,twenty-sevenuniquerestrictionsiteswereevenlydispersedwithanaveragedistancebetweentwoadjacentsiteslessthan50bptofacilitateasystematicinvestigationonstructure-functionalrelationshipofthisproteinbysite-directedmuta-genesis.Tosynthesizeit,thewholegenewasdividedintothreelargefragments(EP,PNandNH)whichwereassembledfromseveralchemicalsyntheticoligonucleotidesbyenzymaticmethod.TheassemblyofboththefragmentEPfromsixoligonucleotides(A-F)andthefragmentPNfromfouroligomers(G-J)wascatalyzedbyT4DNAligaseinusingthesinglestrandedDNAmethod[Chen,H.-B.etal,Nucl.AcidsRes.,18,871(1990)].AndfragmentNHwasformedfromthreeduplexesK,LandMbytheclassicaldoublestrandedDNAmethod.Finally,

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