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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 7 of 17

expression reactions in concentrations from 5 to 80 µM [Supplementary Figure 3]. After expression, the
solubilization of the synthesized MS2-L peptides was monitored via immunoblotting of supernatant and
precipitate fractions. At 80 µM ND concentrations, both constructs were finally solubilized to 80%-100%.
However, there are apparent differences in the solubilization kinetics of the full-length MS2-L toxin and of
the derivative containing a truncated soluble domain. According to the expression rates, the concentrations
of the MS2-L peptides in the CF reactions can roughly be estimated to be 60-100 µM. The truncated
MS2-Lp construct achieved complete solubilization at ND concentrations as low as 20 µM, indicating
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multiple monomer insertions into one ND. In contrast, the full-length toxin MS2-L showed a continuous
ND concentration-dependent solubilization with the highest values at approx. 80 µM NDs. This result gives
the first evidence of a potential negative effect of the soluble domain on the membrane insertion property of
MS2-L. No preference in membrane insertion could be detected for the lipid head group charge
[Supplementary Figure 3]. In further tests with 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)
(DOPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-(1’-rac-glycerol) (POPG) having increased lipid chain
length and lipid flexibility, no difference in membrane insertion, if compared with DMPG NDs, was
detected (data not shown).

DnaJ interaction with MS2-L in NDs and in micelles
The open access of CF reactions allows to synthesize MS2-L in the presence of supplied potentially
interacting proteins and to study effects on functional folding, membrane insertion or stability. Former
[4]
studies suggested an interaction of MS2-L with the chaperone DnaJ . In a related system with the phage
toxin ΦX174-E, we previously demonstrated that interaction with the chaperone SlyD was necessary to
[17]
solubilize the nascent toxin inside the cytoplasm and to promote its membrane insertion . We therefore
analyzed the effects of a potential DnaJ interaction on membrane insertion, assembly and solubility of
MS2-L constructs. To have control over the DnaJ concentration in the CF expression reaction, S30 lysates
[20]
were prepared from the dnaJ negative strain BW25113ΔDnaJ . The DnaJ protein was furthermore
overexpressed in E. coli and purified via its C-terminal His -tag as specified in the methods section.
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First, we analyzed whether DnaJ interaction is necessary for the solubilization and membrane insertion of
MS2-L. The toxin was synthesized either in the P-CF mode or in the presence of NDs (DMPG) in the
BW25113ΔDnaJ lysate in the absence or presence of supplemented purified DnaJ protein [Figure 1A and B].
The solubilization of MS2-L was monitored after CF expression via densitometric analysis of immunoblots
of pellet and supernatant fractions. In the P-CF mode, all synthesized MS2-L protein precipitated,
independent of the presence of supplied DnaJ [Figure 1A]. Furthermore, the co-translational insertion of
MS2-L into provided ND (DMPG) membranes was similarly efficient in the absence or presence of 100 µM
DnaJ [Figure 1B].

In the next approach, possible interactions between MS2-L and DnaJ were analyzed in vitro in CF reactions.
First, purified DnaJ was added to D-CF expression reactions of MS2-L with Brij78 and the toxin was
purified after expression via its StrepII-tag. DnaJ was co-purified with MS2-L from the RM, indicating a
stable toxin/chaperone complex that is formed in detergent either co-translationally or post-translationally
[Figure 1C].

Furthermore, the post-translational interaction of DnaJ with MS2-L derivatives solubilized in either Brij78
detergent or NDs was analyzed in pulldown assays. Full-length MS2-L as well as constructs containing
deletions of the N-terminal soluble domain and resulting in truncations of the first 22 (MS2-Lp ),
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25 (MS2-Lp ), 28 (MS2-Lp ), 31 (MS2-Lp ) and 34 (MS2-Lp ) amino acids were analyzed. The constructs
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35
32
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were CF synthesized in BW25113ΔDnaJ lysate and either inserted into NDs (DMPG) or solubilized in Brij78