Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase 英文参考文献.docVIP

OptimizedExpressionandPurificationforHigh-Activity PreparationsofAlgal[FeFe]-Hydrogenase IftachYacoby1*¤a,LottaTollstoyTegler1,SergiiPochekailov1¤a,ShuguangZhang1*,PaulW.King2* 1Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2Biosciences Center, National RenewableEnergyLaboratory,Golden,Colorado,UnitedStatesofAmerica Abstract Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographicstudies. PrincipalFindings:Wedescribetheconstruction,overexpressionandhigh-yieldpurificationofafusionproteinconsisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized throughimprovementsincultureconditionsandmediacomponentsusedforexpression.Wealsodemonstratedthatfusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinityofFdforDEAEallowedfortwo-steppurificationbyionexchangeandStrepTactinaffinitychromatography. Inaddition,theincorporationofaTEVproteasesiteintheFd-HydA1linkerallowedfortheproteolyticremovalofFdafter DEAEstep,andpurificationofHydA1alonebyStrepTactin.Incombination,thisprocessresultedinHydA1purificationyields of5mgL21ofculturefromE.coliwithspecificactivitiesof1000U(U=1 mmolhydrogenevolvedmg21min21 ). Significance: The [FeFe]-hydrogenases arehighly efficient enzymesand their catalytic sites provide model structures for syntheticeffortstodeveloprobusthydrogenactivationcatalysts.Inordertocharacterizetheirstructure-function