Strains
Two E. coli strains were used in this study: the X-press strain,
a BL21(DE3) derivate patented by enGenes Biotech GmbH (Mairhofer et al.,
2016; Stargardt et al., 2020), and a state-of-the-art BL21(DE3) strain
(New England Biolabs, Ipswich, MA). The X-press strain carries a
genomically integrated sequence coding for Gp2, a protein repressing
cell growth by inhibition of RNA polymerase. Its expression is induced
by L-arabinose, which cannot be degraded by X-press due to a knockout of
the araABCD operon. For determination of cell growth repression
solely induced by Gp2 expression, the plasmid free X-press strain was
used. For recombinant protein production, both strains were transformed
with a pET30a plasmid containing a cer sequence for enhanced
plasmid stability (Bower & Prather, 2009) and a kanamycin resistance
marker. The plasmid carried the gene coding for 1) the IgG-binding
domains of Protein A from Staphylococcus aureus (SpA) with the
pelB signal sequence or 2) the anti-TNFRI VHH single domain antibody
DOM101 with the ompA signal sequence (Chatel et al., 2014). Both
proteins were His-tagged at the C-terminus. Protein sequences are listed
in Supporting Information 1.