Journal of Cell and Molecular Biology - ResearchGate

II SFM were grown in 100 – 500 ml Erlenmeyer
flasks on an orbital shaker at 29ºC. The suspension
culture was infected with the virus carrying the B17
gene when the cells were in mid-exponential
growth and the density of cells is between 1 – 3 x
10 6 cells/ml. 2 plaque-forming units (pfu) were
added to each cell in suspension, a parameter that is
usually called multiplicity of infection, MOI.
Harvesting is done 24 to 48 hours later. The media
is then centrifuged at 250xg for 5 minutes, and the
supernatant is collected and stored in 2 μg/ml
PMSF or aprotinin, 2 mM EDTA, and 0.05% NaN3
at 4 ºC.
The transfection with the B17 gene was done
with a pDLST8 plasmid containing the B17 cDNA
sequence into a recombinant donor plasmid. The
donor plasmid was then hosted for one day in
competent DH10Bac E. coli cells, and
subsequently transposed for antibiotic selection for
2 days in E. coli (Lac7) containing a recombinant
bacmid. In day 4, the recombinant bacmid DNA
was introduced into the Sf-9 cells for recombinant
virus particles to be produced the next two days. A
viral titer was done by plaque assay to determine
the number of pfus in the stock and to concentrate,
if necessary. The viral stock was then used to infect
other suspension cultures.
Aliquots of the stored media stock were
incubated for two hours in glass tubes containing
protein-G Sepharose in the ratio 4:1 to remove
media IgGs prior to the last incubation for two
hours in an immuno-adsorbant column. This
immobilization column contained a similar bed
volume of protein-G sepharose coupled with antiapo
B IgG. The anti-B IgG was crosslinked to the
protein-G Sepharose with DMP in TEA or a basic
Na-phosphate buffer, and the blocking was
achieved with ethanolamine. The immobilized B17
was then eluted with an acidic glycine buffer (pH
2.5), and the eluate was brought a neutral pH by
adding a volume of tris (pH 8) amounting to 10%
of the total elution volume. The eluate was then
tested for purity, dialyzed against a K-phosphate
buffer (pH 7.4) and stored at 4ºC.
The concentration of B17 in solution was
initially determined by the Lowry method using
bovine serum albumin as the standard (Lowry et
al., 1951). Alternatively, a multiplicity factor was
determined so that the concentration can be
approximated from the direct measurement of the
protein UV absorbance at 280 nm, A280. This was
achieved by preparing two sets of samples with the
same amount of protein in each sample. One set
was diluted with differing volumes of chemical
Expression, purification and quantification of B17 39
denaturant (e.g. urea), while the other was diluted
with the same volumes of the sample buffer. The
absorbance A280 was then measured for both sets
and the one with denaturant was compared with the
reported A280 of tryptophan and tyrosine. A
multiplicity factor of 2 was then determined such
that the concentration of B17 in that solution is
equal to A280 x 2 (mg/ml).
Results
Figure 1 shows the initial protein production check
that was done in the early stages of cell growth –
before introduction to roller bottles or bioreactor.
The Western blot shows a single band
corresponding to B17 – probed by the apoB
polyclonal antibody.
Figure 1. B17 production from C-127 cells. The
band corresponding to B17 on (A) Coomassie
stained SDS-PAGE and (B) the Western blot of the
identical gel, aligned with Bio-Rad broad range
protein marker.
The large-scale mass expression in the roller
bottle or the bioreactor, however, showed that, upon
prolonged incubation necessitated by these
techniques, degradation products begin to form
(Figure 2), a problem that was solved by the
addition of a protease inhibitor, PMSF, to the
conditioned media (Figure 3).
Figure 2. Degradation problem in the mammalian
system. Western blot of B17 from the bioreactor
media samples taken at different incubation times
showing the appearance of the degradation product
as a function of time.