Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

Abstract

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl2 induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.

Fig. 1

Western blot analysis of transient and stable expression of rSSL in Drosophila S2 cells. Cells grown in DES medium transfected with the plasmid pMT/BiP_SSL were induced with 500 μM CuSO₄. Fifteen μL of supernatant was harvested after 4 days and screened for rSSL expression by western blotting using the tetra-His antibody after 15 % reducing SDS-PAGE. Transient expression is shown in A. Lanes are (1) 0.7 μg pure E. coli-produced rSSL, (2) fresh medium, (3) non-induced S2 cells, (4–7) supernatants at 1, 2, 3 and 4 days post-induction. Stable clonal cell lines are shown in B, with each lane showing a distinct line. In each panel, molecular mass marker sizes (kDa) are indicated and the arrow indicates the position of the rSSL protein

Fig. 2

Western blot analysis of optimization of inducer concentration for the expression of rSSL in stable transfected Drosophila S2 cells. Cells were grown in serum-free medium and induced with CdCl₂ or CoSO₄. Fifteen μL of supernatant was harvested after 4 days and screened for rSSL expression by western blotting using tetra-His antibody on 15 % reducing SDS-PAGE. Lanes are indicated with inducer concentrations and the lane NC contained negative fresh control medium. The arrow indicates the position of the rSSL protein

Fig. 3

Purification of rSSL from Drosophila cell culture supernatant. Cells were grown in serum free medium induced with 3 μM CdCl₂ and harvested after 4 days. The rSSL was purified by yeast affinity and IMAC and 15 % reducing SDS-PAGE followed by Coomassie staining (A) and western blotting using anti-SSL antibodies (B) were performed with 15 μL of fractions loaded in each lane. For A and B, lanes are (M) molecular mass marker, (1) 10 × concentrated culture supernatant, (2) 10 × concentrated yeast suspension, (3–4) 10 × concentrated TCS washes, (5) mannose eluate concentrated by ultrafiltration, (6) IMAC flow-through; (7–9) 15 μL imidazole washes; (10–11) 15 μL purified rSSL; (12) 15 μL 500 mM imidazole wash. The arrow indicates the position of the rSSL protein

Fig. 4

Determination of rSSL concentration in culture supernatant by western blot analysis. Supernatant was subject to 15 % reducing SDS-PAGE followed by western blotting using anti-SSL antibodies alongside pure native SSL, which was used as a standard. Lanes are (1) 6.25 ng SSL, (2) 31.3 ng SSL, (3) 62.5 ng SSL, (4) 188 ng SSL, (5) 313 ng SSL, (6) 500 ng SSL, (7) 125 ng SSL, (8) Drosophila—expressed rSSL representing 15 μL of supernatant. Molecular mass marker sizes (kDa) are indicated. The arrow indicates the position of the rSSL protein

Fig. 5

Interaction of rSSL with A. salmonicida. Purified rSSL, TCS buffer or fresh insect cell culture medium were incubated with A. salmonicida, bacteria were washed and then bound materials were eluted using mannose. Samples (15 μL per lane) were subject to 15 % reducing SDS-PAGE followed by western blotting using anti-SSL antibodies. Lanes are (1) 15 μL purified rSSL, (2) 15 μL unbound rSSL, (3–4) 15 μL rSSL washes, (5) 15 μL rSSL mannose eluate, (6) 15 μL unbound TCS buffer, (7–8) 15 μL TCS buffer washes, (9) 15 μL TCS buffer mannose eluate, (10) 15 μL unbound culture medium, (11–12) 15 μL culture medium washes, (13) 15 μL culture medium mannose eluate. The arrow indicates cross-reacting rSSL protein

Fig. 6

Oligomerization of Drosophila rSSL. Bacterial and Drosophila cell-expressed rSSL were subject to SDS-PAGE under non-reducing conditions and then western blotting using the tetra-His antibody. Lanes are (1) 0.7 μg purified rSSL expressed in E. coli, (2) 15 μL of purified Drosophila rSSL. Molecular mass marker sizes (kDa) are indicated