Biomedical Engineering Reference
In-Depth Information
was incubated overnight at 37
C in Luria-Bertani (LB) medium containing
100
g/mL ampicillin and agitated at 250 rpm. A volume of 5 mL of the culture
was inoculated into 500 mL medium and induced with 0.5 mM IPTG when the
cell density reached 0.5-0.6 unit at OD600. Cells were harvested 3 h after Isopropyl
b
m
-D-1-thiogalactopyranoside (IPTG) induction by centrifugation at 5,000
g
for
70
C before use.
The Strep-tagged connector was purified by affinity chromatography with Strep-
Tactin (IBA, St. Louis, MO). Cells were resuspended with Buffer W (15%
glycerol, 0.5 M NaCl, 1 mM EDTA, 100 mM Tris-HCl, pH 8.0), and the cleared
lysate was loaded onto a Strep-Tactin Sepharose Column and washed with
Buffer W. The Strep-tagged connector was eluted by buffer E (15% glycerol,
0.5 M NaCl, 1 mM EDTA, 2.5 mM desthiobiotin, 100 mM Tris-HCl, pH 8.0).
The His-tagged connector was purified with Nickel affinity chromatography [
46
]
(Novagen). A plasmid was constructed to express the connector protein by a
two-step PCR [
41
]. The connectors were reengineered at the terminal ends, such
as insertion of a His- or Strep-tag at the C-terminus just downstream of a six glycine
linker for improved affinity tag flexibility [
40
,
42
,
45
,
46
]. The plasmid was then
transformed into the
E. coli
strain HMS174 (DE3) for protein expression. The His-
or Strep-tagged connector was subsequently purified to homogeneity by affinity
chromatography (Novagen, IBA) [
46
].
20 min in a Beckman JS-7.5 rotor and then stored at
4.3 Preparation of Lipid Vesicles Containing
the Reengineered Connector
Attempts to directly incorporate the connector into planar lipid membranes to
serve as a nanopore were unsuccessful. A two step procedure is necessary to enable
the insertion of the connector channel into a lipid bilayer with high efficiency:
reconstitution of the connector into a liposome, followed by vesicle fusion to insert
the connector into a planar lipid membrane.
Analysis of the surface charge of the connector revealed that its central surface
region exhibits slight hydrophobicity compared to the two flanking layers at the
wider and narrow ends, respectively (Fig.
4.1c
)[
15
,
16
]. The C-terminal end of
the connector is located at the wider end domain and embedded inside the viral
procapsid. The hydrophobic layer is in contact with the shell of the procapsid
(Fig.
4.1a
).
Lipid bilayers typically contain a hydrophilic-hydrophobic-hydrophilic architec-
ture. To facilitate the lipid membrane insertion, the three hydrophilic-hydrophobic -
hydrophilic layers of the connector were made more distinct by replacing the 25
residues at the C-terminal domain of gp10 with a basic peptide. The modified gp10
was purified to homogeneity and the protein self-assembled into the dodecameric
structure with similar morphology to the 12-fold symmetric wild type connector,
as observed by AFM, TEM and SDS-Page gel (Fig.
4.2a-d
). The existence of a native
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