2.1.1 Strains
E. coli K-12 NEB5-α [fhuA2Δ(argF-lacZ)U169 phoA gln V44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ] was obtained from New England Biolabs (NEB, Ipswich, USA) and used for all cloning procedures. For recombinant protein expression, linear DNA cartridges controlled by the T7 promoter were integrated into the bacterial chromosome at the attTn7 site of E. coli BL21(DE3) [fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 ] (NEB). Hereafter, this strain is referred to as B3<T7-gene >. Linear DNA cartridges controlled by the A1 promoter were integrated into the bacterial chromosome at the attTn7 site of E. coli BL21 [fhuA2 [lon] ompT gal [dcm] ΔhsdS] (NEB) containing thelacIQ promoter as described by Glascock and Weickert.[25] Briefly, the pETAmp-lacIq plasmid was constructed for integration of the lacIQpromoter in E. coli BL21. This plasmid contains the ampicillin resistance gene flanked by FRT sites and the lacI gene controlled by the lacIQ promoter. The pBR322 ori andlacI were amplified from pET30a using the overhang PCR technique to introduce a C -> T mutation in the lacI promoter. The linear lacIQ DNA cartridge for genome integration was amplified using Q5® High-Fidelity DNA Polymerase (NEB) according to the manufacturer’s manual. Integration into the bacterial chromosome occurred at the lac operon site ofE. coli BL21 carrying the pSIM5 plasmid.[26] This strain was designated as BL21Q and referred to hereafter as BQ <A1-gene >.
The cytosolic protein GFPmut3.1 was used as a recombinant “easy-to-produce” model protein.[27] An antigen-binding fragment (Fab) against TNF-α (FTN2) with the DsbA signal sequence (dFTN2) was used as a recombinant “challenging” model protein.[28]