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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 3 of 17

with SlyD by a combined approach including laser-induced liquid-bead ion desorption (LILBID) mass
[17]
spectrometry, nanodisc (ND) technology and CF expression studies . SlyD forms a stable complex with
the nascent soluble domain of ΦX174-E and presumably prevents the formation of an intramolecular
interaction between the soluble domain and the transmembrane domain that would result in inactive
ΦX174-E. The ΦX174-E/SlyD complex stays soluble and keeps the toxin in a membrane insertion competent
conformation. After membrane insertion, ΦX174-E polymerizes into high-order complexes that are
presumably essential for subsequent lesion formation and cell lysis. Based on the striking similarities of the
MS2-L and ΦX174-E lytic pathways, we now have extended our studies to the membrane insertion
mechanism of MS2-L and complex formation with its interaction partner DnaJ. The possibility to insert
MS2-L derivatives co-translationally into preformed ND membranes in defined CF reaction environments
facilitated an evaluation of the effects of supplied DnaJ.

METHODS
DNA techniques
The coding regions of MS2-L derivatives (MS2-L, MS2-L-GFP, MS2-Lp , MS2-Lp , MS2-Lp , MS2-Lp ,
32
23
29
26
MS2-Lp ) and the dnaJ gene were cloned into the NdeI and XhoI restriction sites of vector pET21a
35
(Novagen). All peptides were modified with either a C-terminal StrepII-tag or a GFP-His fusion separated
10
by a GTGG linker. The full-length constructs MS2-L and MS2-L-GFP contained a codon exchange from
[18]
GAA (Glu) to AAA (Lys) at the first amino acid position following the start codon methionine . The first
ten codons of MS2-Lp and MS2-Lp were optimized for E. coli codon usage to improve expression
29
32
efficiencies. The construct mScarlet-MS2-L was cloned into the vector pET29 (NdeI/XhoI). The construct
contains a GGTG linker between mScarlet and MS2-L and a GG linker between MS2-L and a C-terminal
StrepII-tag.
Cell-free expression reaction
All toxin derivatives were synthesized in a two-compartment continuous exchange cell-free (CF) system
consisting of a reaction mixture (RM) and a feeding mixture (FM) as described previously . As a lysate
[19]
source, S30 lysates either from strain A19 or from the dnaJ deficient strain BW25113ΔDnaJ were used .
[20]
S30 lysate and T7 RNA polymerase were prepared as previously described in detail [21,22] . For all CF
2+
expression reactions, a defined RM to FM ratio of 1:15 was applied and a previously determined Mg
optimum of 20 mM was used for toxin expression. The co-translational solubilization of the synthesized
hydrophobic peptides was achieved either by adding defined concentrations of preformed NDs [lipid-based
cell-free expression (L-CF) mode] or by supplementing the reaction with 0.4% of the detergent Brij78
[detergent-based cell-free expression (D-CF) mode].

Preparation of nanodiscs
NDs were preformed with the MSP1E3D1 scaffold protein and either 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-182 glycerol) (DMPG) lipids.
MSP1E3D1 was expressed and purified as previously published [17,23] . NDs were formed by combining
purified MSP1E3D1 with the selected lipid and 0.1% dodecyl phosphocholine (DPC). The stoichiometry of
MSP1E3D1 to lipid was 1:115 for DMPC and 1:110 for DMPG, respectively. The mixture was incubated for
1 h at room temperature (RT) on a shaking device. Detergent removal was further achieved by subsequent
dialysis against 3 × 5 L of DF buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl) at RT. Each dialysis step was
carried out for at least 12 h. After dialysis, NDs were centrifuged at 22,000 × g and 16 °C for 20 min and
concentrated by ultrafiltration (MWCO 10 kDa) to a concentration of 500-1,000 µM.