Molecular tagging with SNAP-tag
Versatile Molecular tagging of NDRG1 with SNAP-tag
technologyZhen A. Lu
1
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
6
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
11
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
