Expression and purification of the recombinant spike in insect
larvae
A low-cost alternative to cell culture-based protein production is the
use of live insect larvae as “mini bioreactors”. To obtain the
recombinant version of the S protein, we cloned the expression cassette
into the pFastBacDualGFP vector under the control of the strong
baculovirus polyhedrin promoter. The expression cassette included gp64
signal peptide, which targets the recombinant protein to the secretion
pathway. After transfection and amplification in Sf9 insect cells, a
recombinant baculovirus seed-stock for expression in larvae was
obtained. Insect larvae support many of the post-translational
modifications that enable proteins to achieve their biologically
functional native conformation (Loustau et al., 2008).
R. nu larvae were infected by injection of the baculovirus by
intrahemocele injection with approximately 5x105 pfu
of viral stock (Figure 2a-b). From our experience, this dose was the
best option to achieve a high level of S protein expression and no
larval mortality. The larvae infected with the recombinant baculovirus
expressed, in addition to the proteins of interest, the EGFP protein,
which allowed us to determine the optimum day of harvest by observation
under UV light. At 4 dpi, the fluorescence was maximal, and then the
viability of the larvae decreased significantly. Therefore, the larvae
were harvested at 4 dpi under a UV lamp using fluorescence as an
indicator of infection. We proceeded to obtain the clarified homogenate
from the larvae and purify the recombinant proteins expressed in insect
larvae R. nu by IMAC. The gp64 signal peptide was effective to
target S protein to the secretory pathway. The recombinant S protein was
secreted to hemolymph and this localization made it easier to extract it
from the larvae.
SDS-PAGE revealed that most of the proteins of the crude extract were
removed in the passthrough fraction during purification by IMAC when the
sample was loaded without imidazole. However, when the bound material,
containing the protein of interest, was desorbed after a single 500 mM
imidazole step, an important contaminant of hemolymph (hexamerin, 76 kDa
approx.) was still present in the elution fraction. After some rounds of
optimization, we concluded that when the extracts were run directly on
the chromatographic matrix previously equilibrated with 20 mM imidazole,
most contaminants, including hexamerin, eluted in the passthrough or
washing fractions, while the recombinant S protein remained bound to the
matrix. Nevertheless, in this condition, another contaminant with a
molecular weight similar to that of the S protein was present and eluted
with 80 mM imidazole (Figure 3). For these reasons, we assessed a new
protocol, which directly equilibrated the matrix with the same buffer
containing 80 mM imidazole. As judged by reducing SDS-PAGE and WB, the
estimated molecular weight of the recombinant S protein was 150 kDa
(monomer), indicating that it was correctly glycosylated and did not
suffer protease degradation (Figure 3). Other authors have expressed the
ectodomain of S protein with the native peptide sequence in hemolymph ofBombyx mori larvae and reported that recombinant protein was
cleaved probably by a host furin-protease. In the same work, the authors
resolved it with a version of S protein modified in furin
protease-target site (Fujita et al., 2020). In the present work, we
decided to synthesize a version of S protein where the furin cleavage
site (residues 682–685, PRRA) was removed because furin protease
activity was only described in Spodoptera frugiperda , a R.
nu related species (Westenberg et al., 2002).
The amount of recombinant S protein was 15 ug/g of larvae at day 4 p.i.
on our platform based on R. nu . The process based on Bombyx
mori previously reported by other authors (20) can be compared with
ours as follows: in both cases, the optimal day of S protein harvest was
4 dpi after larval infection; however, in Bombyx mori, it was
necessary to extract the whole hemolymph of each larva for purification
while, in our case, a complete extract was done with all infected
larvae, thus simplifying the biotechnological process. In B.
mori , the estimated value of the purified S protein from 10 ml
hemolymph (35 larvae) was 100 µg, and the same yield was obtained with
45 R. nu larvae in the process herein described.