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A monovalent streptavidin with a single femtomolar biotin binding site
Mark Howarth, Daniel J.-F. Chinnapen, Kimberly Gerrow, Pieter C. Dorrestein, Melanie R. Grandy, Neil L. Kelleher, Alaa El-Husseini & Alice Y. Ting
General. Biotin (a gift from Tanabe USA) was dissolved in DMSO at 100 mM. Biotin-adenosine monophosphate (biotin-AMP) was synthesized as described ADDIN REFMGR.CITE Coleman200212RNA-catalyzed thioester synthesisJournal12RNA-catalyzed thioester synthesisColeman,T.M.Huang,F.2002/11Acyl Coenzyme AAdenosineAdenosine MonophosphateBase SequenceCatalysischemistryCoenzyme AKineticsmetabolismMolecular Sequence DataResearch Support,U.S.Gov't,Non-P.H.S.RnaRNA,CatalyticSUBSTRATESulfhydryl CompoundsNot in File12271236Chem.Biol.911Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406, USAPM:12445773Chem.Biol.11 and stored lyophilized at 20C. Aliquots of biotin-AMP were prepared in PBS at 1mM and stored at 20C for use within a month. SDS-PAGE was performed at 200 V with the gel box (X Cell SureLock, Invitrogen) surrounded by ice to prevent dissociation of the streptavidin subunits during electrophoresis.
Plasmid construction. Wild type core streptavidin in pET21a(+) (Novagen) was a kind gift from P.Stayton (University of Washington, Seattle) ADDIN REFMGR.CITE Klumb199810Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complexJournal10Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complexKlumb,L.A.Chu,V.Stayton,P.S.1998/5/26analogs & derivativesanalysisBacterial ProteinsBinding,CompetitiveBiotinCalorimetrychemistryEntropygeneticsHydrogen BondingKineticsmetabolismMutagenesis,Site-DirectedMutationOxygenProtein BindingProteinsResearch Support,U.S.Gov't,P.H.S.StreptavidinThermodynamicsUreaNot in File76577663Biochemistry3721Department of Bioengineering, University of Washington, Seattle 98195, USAPM:9601024Biochemistry12. Mutations in streptavidin were generated by QuikChangeTM (Stratagene) using the following primers and their reverse complements: 5 2 - g g c a c c t g g t a c g c c c a g c t g g g a g a c a c c t t c a t c g t t a c t o i n t r o d u c e N 2 3 A a n d S 2 7 D , 5 2 - t c t g a c c g g t a c c t a c g a a g c c g c t g t t g g t a a c
g c t g a a t t o i n t r o d u c e S 4 5 A , a n d 5 2 - c g c t c a c t c c g c t a t c a c c t g g t c t g g c c t o i n t r o d u c e T 9 0 I . M u t a t i o n s w e r e c o n f i r m e d b y D N A s e q u e n c i n g . T o g e n e r a t e t h e a l i v e s t r e p t a v i d i n s u b u n i t ( A ) , s i x h i s t i d i n e r e s i d u e s w e r e a d d e d t o t h e C t e r m i n u s b y P C R o f s t r e p t a v i d i n u s i n g t h e p r i m e r s 5 2 - t c c a g a a t t c g t a a c t a a c t a a a g g a g a a n d 5 2 - a g a c
a a g c t t t t a t t a a t g g t g g t g a t g g t g a t g g g a a g c a g c g g a c g g t t t . T h i s P C R p r o d u c t w a s c l o n e d i n t o t h e B a mHI and HindIII sites of pET21a(+)-streptavidin ADDIN REFMGR.CITE Klumb199810Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complexJournal10Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complexKlumb,L.A.Chu,V.Stayton,P.S.1998/5/26analogs & derivativesanalysisBacterial ProteinsBinding,CompetitiveBiotinCalorimetrychemistryEntropygeneticsHydrogen BondingKineticsmetabolismMutagenesis,Site-DirectedMutationOxygenProtein BindingProteinsResearch Support,U.S.Gov't,P.H.S.StreptavidinThermodynamicsUreaNot in File76577663Biochemistry3721Department of Bioengineering, University of Washington, Seattle 98195, USAPM:9601024Biochemistry12. AP-neuroligin 1 was generated from pEGFP-G1 containing mouse neuroligin-1 ADDIN REFMGR.CITE Levinson2005135Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificityJournal135Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificityLevinson,J.N.Chery,N.Huang,K.Wong,T.P.Gerrow,K.Kang,R.Prange,O.Wang,Y.T.El Husseini,A.2005/4/29analysisAnimalsBlotting,WesternBrainCells,CulturedchemistryCloning,MolecularcytologyDnaDNA,ComplementaryElectrophysiologyGene LibraryGreen Fluorescent ProteinsHippocampusImage Processing,Computer-AssistedImmunohistochemistryMembrane ProteinsmetabolismMiceMicroscopy,FluorescenceModels,BiologicalMultigene FamilyNerve Tissue ProteinsNeuronsphysiologyPROTEINProtein BindingProteinsRatsRats,WistarRecombinant Fusion ProteinsResearch Support,Non-U.S.Gov'tSynapsesTransfectionNot in File1731217319J.Biol.Chem.28017Department of Psychiatry, the Brain Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, CanadaPM:15723836J.Biol.Chem.13 by replacing GFP with the acceptor peptide (AP) ADDIN REFMGR.CITE Chen2005133Site-specific labeling of cell surface proteins with biophysical probes using biotin ligaseJournal133Site-specific labeling of cell surface proteins with biophysical probes using biotin ligaseChen,I.Howarth,M.Lin,W.Ting,A.Y.2005/2BiotinBiotinylationCarbon-Nitrogen LigasesCell LineCellschemistryEpidermal Growth FactorEscherichia coliEscherichia coli ProteinsFluorescent DyesHela CellsHumansKidneyKineticsLigasesMembrane ProteinsmetabolismmethodsMicroscopy,FluorescenceMolecular Probe TechniquesMOLECULESPROTEINProteinsReceptor,Epidermal Growth FactorRepressor ProteinsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,Non-P.H.S.Research Support,U.S.Gov't,P.H.S.Sensitivity and SpecificityStaining and LabelingSUBSTRATESURFACETranscription FactorsNot in File99104Nat.Methods22Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Room 18-496, Cambridge, Massachusetts 02139, USAPM:1578220 6 <