Biology Reference
In-Depth Information
yields material that can be purified with relative ease and is biologically active (
Tian,
Thomas, & Cowan, 2010
).
11.1.2
Expression of TBCA, TBCB, and TBCC
TBCA, TBCB, and TBCC can be expressed as soluble, biologically active recombi-
nant proteins in good yield in
E. coli
host cells. Note that in the case of TBCC, the
protein exists as two isoforms: short (TBCC) and long (TBCC-L). The biological
significance of these two isoforms is unknown; both are equally active in
in vitro
heterodimer assembly assays. The protocol below is for the expression and purifica-
tion of the long form.
11.1.2.1
Bacterial cell growth and induction
The following protocol is for the preparation of bacterial host cells expressing either
TBCA, TBCB, or TBCC:
a.
Transform the T7 expression plasmid into
E. coli
BL21DE3 cells using antibiotic
selection appropriate for the pET vector in use. Incubate at 37
C until the
resulting bacterial colonies are easily visible to the naked eye (typically
overnight).
b.
Transfer the colonies en masse into a flask containing 1 l of sterile Luria broth
containing the selection antibiotic. This can be conveniently done by pipetting
5 ml of broth directly onto the plate, scraping up the colonies with a sterile
spreader, and transferring the bacterial suspension to the growth flask. It is not
necessary to pick a single colony; indeed, in addition to the greatly extended
time required for growth, doing so may lead to diminished yield because of the
larger number of division cycles with an accompanying enhanced chance of
plasmid loss.
c.
Grow with shaking at 37
C until
A
600
ΒΌ
1.0-1.2. Remove 1 ml of culture as an
uninduced control. Induce expression in the bulk culture by the addition of
isopropyl thiogalactoside to 1 mM. Continue growth with shaking at 37
C for a
further 2.5 h; remove a 1 ml aliquot of the culture for use in determining the
efficiency of expression of the recombinant protein. Harvest the bacteria in these
small aliquots by centrifugation in 1.5 ml Eppendorf tubes, discard the
supernatant, and suspend directly in SDS-PAGE loading buffer. If necessary, the
pellet can be efficiently dispersed by brief sonication using a micro tip powered
by an ultrasonic generator.
d.
Harvest the bulk of the bacterial culture by centrifugation at 10,000
g
.
e.
Resuspend the bacterial pellet in 10 ml 10 mM Tris-HCl, pH 7.2, 1 mM EDTA
(T
10
E
1
) by vigorous vortexing. Dilute to 500 ml with T
10
E
1
and repeat step d. At
this stage, the bacterial pellet may be stored frozen at
20
C.
f.
Analyze the bacterial protein content on a 10% SDS-PAGE gel. In each case, the
recombinant protein should be readily visible as a Coomassie-stained band that is
absent from the corresponding uninduced control.
