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--- purification:expression_and_purification_of_smprmt3_184-553 [2016/10/28 13:57] – marechan
+++ purification:expression_and_purification_of_smprmt3_184-553 [2023/01/20 13:33] (current) – removed marechan
@@ Line 1: / Line 1: @@
-**__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<sup>-1</sup>
-**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<sup>-1</sup> 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). Incubate crude extract with 5 mL of equilibrated Talon<sup>®<sup/> 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).
-**Buffers**
-__Lysis buffer (200 mL):__ 50 mM BTP pH9, 250 mM NaCl, 1 mM TCEP, 0.01% NP40, Complete<sup>®</sup>-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