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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 200 mL Lysis buffer
Buffers
Lysis buffer: 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: 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 10 mM imidazole
Talon elution buffer: 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 150 mM imidazole
Dilution buffer: 50 mM BTP pH9, 1 mM TCEP, 1 mM EDTA
ANX buffer: 20 mM BTP pH9