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.