The MBP-HttQ44-exon1-S112C-histag
protein was generated and purified with some modifications from previously
published protocols.9 (link),14 (link),33 (link) The MBP-Htt-ex1 protein was expressed in BL21 E. coli that were grown to an OD600 of 0.6–0.8 and autoinduced.
The bacteria were harvested by centrifugation at 4000 RPM (4 °C,
20 min) and resuspended in a lysis buffer (25 mM HEPES-KOH pH 7.4,
100 mM NaCl, 0.5% Triton X-100, 15 mM imidazole, 10% glycerol, 0.5
mM PMSF, and 5 mM β-mercaptoethanol). Following lysis by French
Press and clarification in the centrifuge at 12 000 RPM (4
°C, 45 min), the protein underwent a first purification by way
of the C-terminal His-tag on a Ni-Sepharose column. The MBP tag was
then utilized to purify the protein on an amylose column, and the
protein was concentrated and exchanged into buffer C (25 mM HEPES-KOH
pH 7.4, 100 mM NaCl, 10% glycerol (v/v)). The MBP-Htt-ex1Q44-exon1-S112C-histag
protein was labeled with Alexa Fluor 647 maleimide (Life Technologies)
and Cy3B maleimide (GE Healthcare) according to our published protocol.33 (link) The dye:protein labeling ratio (0.6–0.7
for Alexa 647 and Cy3B) was used to determine the volumes of protein
needed to prepare the MBP-Htt-ex1:MBP-Htt-ex1-dye = 10:1 ratio (i.e.,
the unlabeled MBP-Htt-ex1 concentration included the unlabeled protein
within the MBP-Htt-ex1-Alexa 647 mixture at 0.6–0.7 labeling
ratios).
protein was generated and purified with some modifications from previously
published protocols.9 (link),14 (link),33 (link) The MBP-Htt-ex1 protein was expressed in BL21 E. coli that were grown to an OD600 of 0.6–0.8 and autoinduced.
The bacteria were harvested by centrifugation at 4000 RPM (4 °C,
20 min) and resuspended in a lysis buffer (25 mM HEPES-KOH pH 7.4,
100 mM NaCl, 0.5% Triton X-100, 15 mM imidazole, 10% glycerol, 0.5
mM PMSF, and 5 mM β-mercaptoethanol). Following lysis by French
Press and clarification in the centrifuge at 12 000 RPM (4
°C, 45 min), the protein underwent a first purification by way
of the C-terminal His-tag on a Ni-Sepharose column. The MBP tag was
then utilized to purify the protein on an amylose column, and the
protein was concentrated and exchanged into buffer C (25 mM HEPES-KOH
pH 7.4, 100 mM NaCl, 10% glycerol (v/v)). The MBP-Htt-ex1Q44-exon1-S112C-histag
protein was labeled with Alexa Fluor 647 maleimide (Life Technologies)
and Cy3B maleimide (GE Healthcare) according to our published protocol.33 (link) The dye:protein labeling ratio (0.6–0.7
for Alexa 647 and Cy3B) was used to determine the volumes of protein
needed to prepare the MBP-Htt-ex1:MBP-Htt-ex1-dye = 10:1 ratio (i.e.,
the unlabeled MBP-Htt-ex1 concentration included the unlabeled protein
within the MBP-Htt-ex1-Alexa 647 mixture at 0.6–0.7 labeling
ratios).
