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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)
1/ 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).
2/ Incubate crude extract with 5 mL of equilibrated Talon® metal-affinity resin for 2h, 4°C under soft agitation. Spin down beads (233G, 2 minutes, 4°C) and discard supernatant. store 5 μL for SDS-PAGE analysis. resuspend beads in 10 mL cold Talon wash buffer and load them on a clean column. Wash the resin with 40 mL Talon wash buffer (Total = 50 mL = 10V column). store 10 μL for SDS-PAGE analysis. In the column, incubate the resin in 7 mL cold Talon elution buffer, 15 minutes, 4°C under soft agitation, then store the eluate on ice. Repeat this step once then pass the remaining buffer through the column to get remaining eluate. store 5 μL for SDS-PAGE analysis. Regenerate the column with 20 mM MES pH5, 300 mM NaCl. store 10 μL for SDS-PAGE analysis.
3/
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