Molecular Tagging of NDRG1 using SNAP‐tag Technology

Abstract

BackgroundSNAP‐tag is a 20kDa versatile protein tag that can be linked to any nucleotide sequence. When fused to a protein, the SNAP‐tag acts as a molecular linker to an array of molecules such as fluorescence probes, biotin, and synthetic compounds. An immediate advantage of the SNAP‐tag as opposed to traditional fluorescence proteins like GFP is temporal control, allowing the user to control the timing of fluorescence expression. SNAP‐tag was fused to NDRG1, N‐myc Down Regulated Gene 1, with the intent to study its localization, expression, and function. NDRG1 is a protein with putative involvement in metastasis suppression, endosomal trafficking, and interaction with surface molecule E‐Cadherin, an important player in mesenchymal to epithelial transition. NDRG1 has been of interest because of its involvement in prostate and breast cancer metastasis.PurposeTo create a NDRG1 and SNAP‐tag fusion protein for use in further study of localization and function of NDRG1MethodsTwo unique SNAP‐tag and NDRG1 fusion sequences, with SNAP at the N terminal in one and at the C terminal in the other, were created, verified, ligated into a hCMV promoter driven pLVx vector, and then transfected into HEK293 cells using Lipofectamine 2000. Cells were incubated in various concentrations of TMR‐Star Red Fluorophore at 37C 5%CO2 for 30 minutes or 1 hour with a parallel control arm of cells transfected with empty vector but were incubated in identical substrate concoctions. The transfected cells, after incubation with the SNAP fluorophore substrate, were verified to express the plasmid and then imaged using Confocal Microscopy. This batch of cells expressing the fusion protein will be used to create a stable cell line, and will be used later on for further study of NDRG1.ResultsThe two constructs (SNAP‐tag attached at the N and C terminals of NDRG1), when transfected into HEK293 cells and subsequently incubated in Optimem + fluorescence TMR‐StarRed substrate achieved equal fluorescence intensity. 1uM of TMR‐Star substrate incubated in 37C and 5%CO2 for 1 hour achieved optimal signal to background ratio and allowed for good imaging conditions. NDRG1‐SNAP expression is seen in the cytoplasm, nucleus, and at the membranes. Intensity is greater in the cytoplasm, but there remain compartments that are void of any fluorescence. Furthermore, faint interactions with cytoskeleton, maybe actin, can be seen. (time lapse video and z‐slices of cells in vitro attached)ConclusionsThe NDRG1‐SNAPtag construct has been successfully synthesized and is able to be viably transfected and subsequently used to study the protein of interest, NDRG1, within HEK293 cells. In this case, fluorescence expression upon incubation of transfected cells with TMR‐StarRed substrate allowed for live cell visualization in vitro, but other small molecules may be conjugated to the SNAP‐tag‐NDRG1 fusion protein in future work. Furthermore, a stable cell line with the fusion sequence integrated pseudo‐randomly into the genome can be generated to study the cell in more in vivo like conditions. Some remaining questions yet to be elucidated are the effect that the SNAP‐tag has on intrinsic NDRG1 function, localization of NDRG1, and on general cell behavior and survival.Support or Funding InformationNCINDRG1 ‐ SNAP nucleotide segment on gel electrophoresis. Approximately 1.7 kb longFigure 1NRDG1‐SNAPtag fusion protein expressed in HEK293T cells. Viewed under fluorescence confocal microscopy.Figure 2