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

“…The histone deacetylase inhibitor sodium butyrate inhibits chromatin condensation and thus promotes transcription in less active regions of the genome (Dorner et al 1989;Chen et al 2002;Li and Li 2006;Zhao et al 2006). Protein expression in rS2 cells is thus enhanced if the transgene is integrated into silent regions of the genome (Chang et al 2002;Lemos et al 2009). However, our results show that this effect is negligible if the insert is located in an active genomic region, as it is the case for highly productive single-cell clones.…”

“…The experimental design and statistical evaluation is described in more detail in the ''Appendix 2''. In addition to the experimental design, several additives that may enhance protein production in rS2 cells were also evaluated, namely DMSO (1 or 5%), glycerol (1 or 5%), sodium butyrate (5 or 10 mM), and the presence or absence of 2 lL/mL Carl Roth His-tag inhibitor cocktail (Jeon et al 2012;Lemos et al 2009;Swiech et al 2008;Chang et al 2002;Park et al 2002). The laboratory-scale expression of recombinant GmGlv was achieved in a 2-L Labfors bioreactor (1-L working volume, Infors HT, Einsbach, Germany) equipped with one pitched-blade impeller (3 9 45°, d = 65 mm) and standard sensors for temperature, pH and oxygen saturation.…”

“…However, our results show that this effect is negligible if the insert is located in an active genomic region, as it is the case for highly productive single-cell clones. Other common additives include DMSO (Chang et al 2002;Jeon et al 2012) and glycerol (Swiech et al 2008). Both substances have been shown to stabilize proteins and act as chemical chaperones during folding (Yoshida et al 2002).…”

“…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…”

Knockout of the caspase 8‐associated protein 2 gene improves recombinant protein expression in HEK293 cells through up‐regulation of the cyclin‐dependent kinase inhibitor 2A gene

“…Strategies for enhancing recombinant protein production based on growth arrest include overexpression of cyclindependent kinase inhibitors (p21 Cip1 or p27 Kip1 ;Bi et al, 2004; T A B L E 1 Summary of raw and processed RNA-seq reads generated from CASP8AP2 mutant clone, 892-7, and parental RNA samples F I G U R E 5 Pathway ranking based on Z-score following enrichment analysis of the differentially expressed genes between the CASP8AP2 knockout and the parental cell lines. Gray bars represent pathways involved in the cell cycle Carvalhal et al, 2003;Fussenegger et al, 1997;Meents et al, 2002), the addition of cytostatic chemical agents such as sodium butyrate or dimethyl sulfoxide (Hendrick et al, 2001;Hunt et al, 2002;Hwa Chang et al, 2002;Liu et al, 2001), and maintaining mild hypothermic culture conditions (Ahn et al, 2008;Fox et al, 2004;Nam et al, 2008). However, improved specific production using these approaches is associated with reduced cell growth and requires a careful balancing of growth with protein expression to achieve overall improvements in recombinant protein yield (Sunley & Butler, 2010).…”