Preparation of FlexiHaloTag clones

     We prepared the FlexiHaloTag clones by transferring the ORF sequences from the Flexi clones into a pFN21A vector (Fig.h-1) using Flexi cloning technology. The HaloTag (animation) is a 33-kDa mutant haloalkane dehalogenase that can rapidly form a stable covalent bond to various ligands composed of aliphatic chloride containing a labeling moiety, such as fluorophores, biotin or sepharose beads 6. pFN21A is a mammalian expression vector that can produce N-terminally HaloTag-fused recombinant proteins.

     In addition to confirmation of the insert size by agarose gel electrophoresis and of both the end sequences of cloned ORF by single-pass sequencing (Fig.h-2), we are also evaluating the production and size of proteins derived from pFN21A ORF clones in HEK293 cells, though these are transiently expressed as N-terminal HaloTag-fusion proteins. Subcellular localization of HaloTag-fusions was observed in HEK293 by HaloTag TMR ligand labeling using fluorescent microscopy (Fig.h-3, left panel). Then, the cell lysates were applied to SDS-PAGE to estimate their molecular masses by fluorescent imaging analysis (Fig.h-3, right panel). Because the N-terminal tag might cause incorrect subcellular localization of certain proteins, it is recommended to transfer the ORF sequence into an appropriate Flexi Vector if needed.

Fig.h-1 pFN21A expression vector Fig.h-2 Confirmation of cloned ORFs Fig.h-3 Expression of N-terminal HaloTag-fusion proteins in HEK293

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