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 CdCl 2 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.

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells

“…Monoclonal cell lines for efficient protein production were prepared by limiting dilution as previously described (Uribe et al 2013;Wang et al 2012;Scotter et al 2006). The method is based on the co-cultivation of single transformants with untransfected feeder cells, followed by antibiotic selection of the clones.…”

“…b Optical density after 10 h growth in GmGlv-containing LB medium as function of the GmGlv concentration cell population is often heterogeneous and the most productive clones become outnumbered by the lessproductive but faster-growing majority (Cherbas and Cherbas 2007). The isolation of single-cell clones is therefore an important step in early process optimization, and is often achieved by limiting dilution (Wang et al 2012;Uribe et al 2013). However, for insect cells no data are available thus far that show a comprehensive comparison between different clones and the polyclonal parental culture.…”

“…Although Cd 2? is a potent inducer (Li et al 2012b;Yang et al 2012;Wang et al 2012;Uribe et al 2013) it also activates endogenous heat-shock promoters (Bunch et al 1988) and is highly toxic to humans, a reason why this substance was not used routinely in the past. The evaluation of 37 publications dealing with the expression of proteins in rS2 cells under the control of the Mt promoter revealed that the majority of authors (n = 24, 65%) used CuSO 4 at concentrations of 400-600 lM, with smaller numbers using higher (n = 6, 16%) or lower concentrations (n = 3, 8%) of CuSO 4 and 11% (n = 4) used CdCl 2 (''Appendix 4'').…”

“…As a concrete example, a spider peptide toxin (4.7 kDa) was expressed in recombinant S2-cells at the 12-L scale and yielded 0.48 mg/L after purification (Escoubas et al 2003). The recombinant expression of a bacteria binding C-type lectin (*16 kDa) in rS2-cells resulted in 1.5 mg/L (Uribe et al 2013), while another C-type lectin-like protein (14-17 kDa) was expressed at around 95 mg/L (Scotter et al 2006). The latter is an example of rS2 cells surpassing all previous attempts to produce this protein in E. coli, baculovirus-infected fall armyworm cells or P. pastoris (Scotter et al 2006).…”

“…CuSO 4 \ 400 lM 3 8.1 Deml et al (1999), Valle et al (2001), Lieberman et al (2007) CuSO 4 400-600 lM 24 64.8 Bernard et al (1994), Nilsen and Castellino (1999), Park et al (1999Park et al ( , 2008, Shin and Cha (2002), Friry et al (2002), Chang et al (2002), , Schetz et al (2003), , Cho et al (2004), Lim and Cha (2006), Johansson et al (2007), Lee et al (2007Lee et al ( , 2009Lee et al ( , 2011, Kim et al (2008) Dubreuil et al (1996), Dubreuil and Grushko (1999), Perret et al (2003), Santos et al (2007) CuSO 4 [ 800 lM 2 5.4 Bunch et al (1988), Graziano et al (1998) CdCl 2 1-100 lM 4 10.8 Uribe et al (2013), Li et al (2012b), Yang et al (2012), Wang et al (2012) but active against the rough mutants E. coli D21 (Ra-LPS), E. coli D21e7 (Rc-LPS), E. coli D21f1 (Rd-LPS) and E. coli D21f2 (Re-LPS) (Yi et al 2013). Furthermore, TnGlv (Lundström et al 2002) and…”