Molecular tagging with SNAP-tag Final (3)

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Molecular tagging with SNAP-tag

Versatile Molecular tagging of NDRG1 with SNAP-tag technologyZhen A. Lu

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The Origin of SNAP-tag

Engineered mutant derived from constitutively expressed hAGT in bacterial and mammalian cells and overexpressed upon Attack of Alkylating Agents.Methylated guanine binds to thimine1 to 1 Suicide enzyme2

SNAP-tag engineered to recognize benzyl-guanine(BG) and benzyl-chloropyrimidine(BC) derivatives

182 residues, ~20kDa

SNAP-tag is directed mutant of hAGTLast ~30 residues truncated with minimal loss of functionNo inherent florescence, must be conjugated with fluorophore 3

Advantages of SNAP-tagTemporal ControlFlexible linkerSpecificity Limit interference on protein function

Flexible linker- versatilitydifferent probes, pull-downs, protein-protein interactions ~20kDa protein, fairly small4

NDRG1394 residue member of N-Myc Down Regulated Gene familyMainly cytoplasmic protein with localization to nucleus and membranesPutative involvement in:Endosomal TraffickingMetastasis SuppressionStress Response/autophagy

Interaction with E-cadherin- known tumor/metastasis suppressor, involved with EMT5

NDRG1-SNAP ConstructN and C terminal fusions, ~1.7 kb longCreated via stitch PCR

N C 3kb1kb2kb

Expressed in lentiviral vector driven by hCMV promoter. Inserted at XhoI and NotI restriction sitesComplementary 5 overhangs. Restriction sites engineered on the ends of the full stitch product

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AcGFP spliced out, SNAP-NDRG1 spliced in7

SNAP TMR*-Star SubstrateAbsorption 554nm, emission 580nmTransfected cells incubated with varying concentrations and timesFollowed by rigorous wash

*Tetramethylrhodamine

Nucleophilic thiol group attacks C electrophile.Concentration and incubation times do not need to be precisely analyzed. Important items: signal to background ratio and specificity, and effect on protein function9

N 1uM 1hr 30f, .3sec

NDRG1-SNAP is overexpressedthat is explanation for overall strong intensity10

C 1uM 1hr 23slices

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Future DirectionsEffect of SNAP moiety on NDRG1 function? Stable cell line how would it look?Conditional localization of NDRG1Molecular interactions of NDRG1

Special ThanksJanet Mendonca Sushant KachhapGail VoelkerCUPID/STOP

ReferencesJuillerat, Alexandre et al. Chemistry & Biology , Volume 10 , Issue 4 , 313 317.Kachhap SK, Faith D, Qian DZ et al. (2007).Curr Protoc Protein Sci. 2013 Sep 24; 73: 30.1.130.1.16.July 6, 2004vol. 101no. 279955-9959 PNAS