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Expression & purification of 6his-SmPRMT3(1-553)
Construct
MGSSHHHHHHSSGTGSGENLYFQGHMSPCDQSSNYDLDMEPNDSSYFGSYGHFEIHGEMINDRVRTESYVNFILSNAEKYFKHKIILDVGSGSGILSIIAAQAGASHVYGVEAADEIYAASHETLRVNNLLERVTFIHGQAESVELPVKKVDVIISEWMGYFLFFESMLDSVLKMASKYLSRDGHIFPRHYTLNLLGVQCSEQLRKRRLEHWNNVYGYNMPALRRAALSEAHVLNLTNEHVTPPISPITILTQSFELVALDLDDMHRNRIYNLSNHCSLLCEQKFHLTIQPTTDINNNSSSSSYELDAIVGYFDVRFDDDADCKVEFSTSPTTPLTHWKQTLLFLDKPIRVKPGDKISGIITIRRATTDNRGLEINLLIGETENSLEIKQTFDLIG
Length = 396 AA
Molecular weight = 44782.3 Da
pI 5.66
ξ(red) 40340 L.M-1
Expresion
Grow pnEAvH_SmPRMT3-184 BL21(DE3) transformants on an LB-Amp plate (1 plate/L culture), overnight 37°C. Resuspend cells in LB medium (5 mL/plate) and innoculate liquide 2X LB medium + 100 μg/mL-1 Ampicilline. Grow culture(s) at 37°C, 200 rpm and measure OD600 every 20 minutes. When OD600 reaches 0.4, set temperature to 20°C. When OD600 reaches 0.6 to 0.8, induce 6his-SmPRMT3(184-553) with 1 mM IPTG and leave the cultures to express the protein overnight.
Purification (for 3L)
Pellet cells by centrifugation (4000 rpm, 45 minutes), then resuspend pellets in cold 200 mL Lysis buffer. On ice, sonicate cell suspension 3 times for 5 minutes (amplitude 60, frequency 0.5), then clarify the lysate (41657G, 45 minutes, 4°C).
Buffers
Lysis buffer (200 mL): 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 0.01% NP40, Complete®-EDTA free protease inhibitor cocktail (1 pill / 50 mL)
Talon wash buffer (100 mL): 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 10 mM imidazole
Talon elution buffer (20 mL): 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 150 mM imidazole
Dilution buffer (180 mL): 20 mM BTP pH9, 1 mM TCEP, 1 mM EDTA
ANX buffer (200 mL): 20 mM BTP pH9, 25 mM NaCl, 1 mM TCEP, 1 mM EDTA
Gradient buffer (100 mL): 20 mM BTP pH9, 1 M NaCl, 1 mM TCEP, 1 mM EDTA
GF buffer (350 mL): 20 mM BTP pH9, 50 mM NaCl, 1 mM TCEP, 1 mM EDTA