E coli shuffle cells

Full-length ChiA (ChiA-FL; residues 1–762), minus the N-terminal periplasmic signal sequence, the ChiA N-terminal region (ChiA-NT; residues 1–417), the ChiA N1-domain (ChiA-N1; residues 1–140), the ChiA N2-domain (ChiA-N2; residues 138–299), the ChiA N3-domain (ChiA-N3; residues 285–417) the ChiA C-domain (ChiA-CTD; residues 419–762) and NttE (residues 1–269) were amplified from the genomic DNA of L. pneumophila strain 130b and cloned into the N-terminal His₆-tagged vector pET-46 Ek/LIC (S4 Table). SslE (residues 67–1497), minus the N-terminal periplasmic signal sequence and mature SslE N-terminal proline-rich region, were amplified from the genomic DNA of E. coli strain W and cloned into the C-terminal His₆-tagged vector pOPINE (S4 Table). These were transformed into E. coli SHuffle cells (New England Biolabs) and grown at 37°C in LB media with 100 μg/ml ampicillin. Expression was induced with 1 mM isopropyl-d-1-thiogalactopyranoside (IPTG) at an OD_600nm of 0.6 and cells were harvested after growth overnight at 18°C. Cells were resuspended in 20 mM Tris–HCl pH 8, 200 mM NaCl, 5 mM MgCl₂, 1 mg/ml DNase I, 5 mg/ml lysozyme, lysed by sonication and purified using nickel affinity chromatography. All samples were then gel filtered using a Superdex 200 column (GE Healthcare) equilibrated in 20 mM Tris–HCl pH 8, 200 mM NaCl.

Nanobody fusion proteins were expressed in E. coli SHuffle cells (New England BioLabs) at 20°C overnight after induction with 0.1mM IPTG. His6-MBP tagged fusion proteins were purified by Ni-NTA (QIAGEN) affinity chromatography and dialyzed overnight in 50mM Tris HCl pH7.5, 150 mM NaCl, 0.5mM TCEP buffer. To remove the His6-MBP tag, fusion proteins were incubated with TEV protease, followed by Ni-NTA affinity chromatography to remove any uncleaved His6-MBP tagged proteins, free His6-MBP tag and TEV protease (also His6-tagged). Purified untagged Nanobody fusion proteins were then dialyzed against 50 mM Tris HCl pH 7.5, 150 mM NaCl 0.5 mM TCEP further purified by gel filtration (Superdex75) and flash-frozen in liquid nitrogen prior to storage at −80°C.

The sE7 scFv was cloned into pET28b with a C-terminal StrepII tag using NcoI and NotI restriction sites. The protein was expressed in E. coli Shuffle cells (New England BioLabs) upon induction with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) in Terrific Broth (TB) medium overnight at 25 °C, 200 rpm. Soluble proteins were extracted into PBS and purified on a prepacked 5 mL StrepTrap column (Cytiva) preequilibrated with PBS. The column was washed with PBS to remove non-specifically bound proteins, and scFvs were eluted using PBS supplemented with 2.5 mM desthiobiotin (Sigma-Aldrich). Elution fractions were concentrated, and the buffer was exchanged to PBS using Amicon Ultra Centrifugal Filters with a molecular weight cutoff of 10 kDa.