TECHNICAL NOTE
7
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protein stability at the small-scale stage, without the need for scale-up or purification.
Materials and Methods
E. coli BL21 (DE3) cells harboring the pET24-6xHis-HRV3C plasmid were grown to an
optical density of A600 = 1.6 in terrific broth (TB) medium at 20 °C unless stated otherwise.
Protein expression was induced by addition of 0.5 mM IPTG overnight at 20 °C unless
stated otherwise.
In addition to E. coli BL21 (DE3) (called strain 1 in the main text), two other strains were
used for finding the optimal expression host strain: Rosetta2 (DE3) (called strain 2 in
the main text), a strain with enhanced translational efficiency for eukaryotic proteins
and Rosetta2 (DE3) pLysS (called strain 3 in the main text), a strain with tight expression
control to suppress protein toxicity.
Cells from a 4 mL culture were harvested by centrifugation and resuspended in lysis buffer
(50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.01% (v/v) 1-thioglycerol, 10 mM
MgCl
2
, 10 µg/mL DNase I, 0.2% (v/v) NP-40, 1 mg/mL lysozyme). Lysis was performed by
repeated freezing and thawing of the cell suspensions.
Following lysis, cell lysates were clarified by centrifugation for 5 min at 20,000 x g at 4 °C to
remove cell debris and insoluble material.
Clarified lysates containing expressed His-tagged HRV3C protease were labeled with the
Andromeda His-Tag Labeling Kit (Cat# AN-030002, NanoTemper Technologies) following
the instructions supplied by the manufacturer. A er labeling, samples were loaded into
Andromeda Capillaries (Cat# AN-041001, NanoTemper Technologies). Loaded capillaries
were placed on Andromeda (NanoTemper Technologies) and subjected to a thermal
ramp from 20 °C to 95 °C with a heating rate of 7.0 °C/min. Analysis was performed in
technical quadruplicates for each condition and data was processed with AN.Stability
