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.
