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Page 4 of 17 Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28

DnaJ expression and purification
For overnight starter cultures, 200 mL LB medium supplemented with 100 µg/mL ampicillin was inoculated
with E. coli T7 Express cells transformed with pET21-dnaJ. 10 L LB medium supplemented with 100 µg/mL
ampicillin and 100 mM glucose were then inoculated 1:100 and incubated at 37 C and 180 rpm. Cells were
o
grown until OD reached 0.6 and protein expression was induced with the addition of 1 mM isopropyl-β-
600
D-thiogalactopyranoside (IPTG). The cells were further grown for 3 h at 37 °C, harvested by centrifugation
(4,500 × g, 4 °C, 30 min) and stored at -80 °C until further use. For purification, the cell pellet of a 10 L
expression was resuspended in 50 mL DnaJ buffer (50 mM Tris-HCl pH 7.5, 400 mM NaCl, 10% (v/v)
glycerol, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 0.1% Triton X-100) containing 1×
complete protease inhibitor cocktail and disrupted by sonication (sonication steps of 3 × 1 min and 3 × 45 s
with cooling intervals of 1 min in between each step). After centrifugation (30,000 × g, 4 °C, 30 min), the
2+
lysate was filtered (0.45 µm) and applied on Ni -NTA agarose (3 mL beads) pre-equilibrated with 5 column
volumes (cv) of DnaJ buffer. Unbound protein was removed by washing with 5 cv of DnaJ buffer and 5 cv
of DnaJ buffer containing 50 mM imidazole. DnaJ was finally eluted in 2 cv of DnaJ buffer supplemented
with 300 mM imidazole. Protein-containing fractions were concentrated by ultrafiltration (MWCO
10 kDa), flash-frozen in liquid nitrogen and stored at -80 °C until further use. Since DnaJ exhibited
sensitivity against lower temperatures, all purification steps were performed at RT.

Purification of MS2-L derivatives
CF expressed MS2-L derivatives were purified by affinity chromatography via their StrepII-tag. After
expression, the RM was centrifuged (14,000 × g, 4 °C, 10 min) and the supernatant was diluted 1:3 in Strep
buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl). When samples were expressed in the D-CF mode, the
working buffer was supplemented with 0.02 % Brij78. 100 µL StrepII-Tactin resin was used for 150 µL of RM
and equilibrated with 5 cv of Strep buffer. The diluted supernatant was applied on the resin and the flow-
through was re-applied three times to achieve optimal protein binding. After washing with 5 cv Strep buffer,
proteins were eluted with 2 cv Strep buffer containing 15 mM d-desthiobiotin. Samples were subsequently
concentrated by ultrafiltration (MWCO 10 kDa) and used for further analysis.

Tris-Tricine SDS-PAGE and immunoblotting
Precipitate forming cell-free expression (P-CF) expressed proteins were resuspended in an S30 buffer C
[10 mM Tris-acetate pH 8.2, 14 mM Mg(OAc) , 60 mM KOAc] in a volume corresponding to the RM
2
volume. All protein samples were supplemented with 1× SDS loading buffer (4×: 100 mM Tris-HCl pH 6.8,
8 M urea, 20% (w/v) SDS, 20% (v/v) β-mercaptoethanol, 15% (v/v) glycerol, 0.12% (w/v) bromphenol blue),
incubated at RT for 10 min and separated using denaturing, discontinuous 4%-11% Tris-Tricine SDS-PAGE
as described earlier . Separation was carried out at 90 V for 10 min and 150 V for another 45 min.
[24]
Subsequently, the gels were either fixated in fixing solution [50% (v/v) ethanol, 10% (v/v) acetic acid] and
stained with colloidal Coomassie Brilliant Blue G250 staining solution [0.02% (w/v) Coomassie Brilliant
Blue G-250, 5% (w/v) aluminium sulfate-(14-18)-hydrate, 10% (v/v) ethanol, 2% (v/v) ortho-phosphoric-
acid] or used for immunodetection via Western Blotting. Following SDS-PAGE, proteins were transferred
onto a methanol activated 0.45 µm PVDF membrane (340 mA, 35 min) in Towbin buffer [25 mM Tris-HCl
pH 8.3, 192 mM glycine, 15% (v/v) methanol]. After protein transfer, the membrane was blocked for 1 h at
RT in 4% (w/v) skim milk powder in phosphate-buffered saline with Tween 20 (PBS-T) [2.6 mM KCl,
1.8 mM KH PO4, 137 mM NaCl, 10 mM Na HPO , 0.05% (v/v) Tween-20] and incubated with the 1st
4
2
2
antibody (α-StrepII-HRP: 1:7,000 dilution in 0.5% skim milk powder in PBS-T or α-His: 1:2,000 dilution)
under the same conditions. In the case of the α-His antibody, the membrane was washed three times with
PBS-T and incubated with α-mouse-HRP (1:5,000 dilution in 0.5% skim milk powder in PBS-T) for 1 h at
RT or at 4 °C overnight. After incubation, the membrane was washed three times with PBS-T and analyzed
by chemiluminescence detection.