Biomedical Engineering Reference
In-Depth Information
the start codon. Purifi cation of GCC185 highly relies on affi nity
chromatography using anti-FLAG resin and a size exclusion step to
remove eluting FLAG peptides and residual contaminants. The
N-terminal GFP can be used for multiple purposes: (1) as a fl uo-
rescent marker to follow protein expression in cells or to detect the
protein molecules in fl uorescence-based assays, (2) as a globular
marker to identify protein's orientation in high-resolution micro-
scopic techniques, and (3) as an affi nity tag for immobilization on
resins to capture binding partner(s).
Cellular characterization of the tethering function of GCC185
is based on its role in the retrograde transport of the mannose
6-phosphate receptors (MPRs) from late endosomes to the
trans-
-
Golgi network (TGN) [
10
,
11
]. MPRs bind newly synthesized
lysosomal hydrolases in the Golgi complex and deliver them to late
endosomes. After releasing these enzymes, MPRs are recycled back
to the TGN [
12
]. The cellular distribution of MPRs can be assessed
by immunofl uorescence microscopic staining using anti-cation-
independent MPR antibody. Normally, at steady state, MPRs are in
perinuclear late endosome and to a lesser extent in the TGN [
13
].
When the tethering is impaired, MPR staining is much more dis-
persed and distributed in cell periphery. This lab has shown that
GCC185 is required for the transport of MPRs from late endo-
somes to the TGN: in the absence of GCC185, MPRs accumulate
in peripheral Rab9
+
and AP-1
+
vesicles [
14
-
16
]. In this manner,
the functionality of a tethering protein and its mutants can be
examined by plasmid rescue after siRNA-protein depletion, in this
case using immunofl uorescence microscopic staining of MPRs to
determine their intracellular localization.
2
Materials
1. Orbital shaker housed in a 37 °C incubator with a humidifi ed
atmosphere supplied with 5 % CO
2
.
2. Erlenmeyer cell culture fl asks or glass fl asks (
see
Note 1
).
3. FreeStyle™ 293-F cells and FreeStyle™ 293 Expression
Medium (GIBCO, Grand Island, NY).
4. Expression constructs: Ligate the cDNA encoding the tethering
protein of interest (here we use human GCC185 full length and
its N-terminal half (residues 1-889)) in the EGFP-C1 plasmid;
insert a FLAG-tag (DYKDDDDK) downstream of the GFP
fl anked by the GGATCC linker (AS) for purifi cation purposes.
2.1 Protein
Expression
in Mammalian Cells
in Suspension
5. Opti-MEM
®
I Reduced Serum Media (GIBCO) or other
medium without serum.
6. Polyethylenimine (PEI) (Polysciences Inc., Warrington, PA):
1 mg/ml PEI pH 7.4. Dissolve 50 mg of PEI in 40 ml sterile
water. Mix the solution on a magnetic stirrer at 50-60 °C until
