u/Danktank452

So I recently started a postdoc and have been tasked with purifying a microsomal P450 enzyme. I did purification of his tagged heme proteins during my PhD and ever had any trouble. This P450 will not stick to the column no matter what I do.

Here is a brief explanation of my protocol and what I have tried. I grow the cells in TB media and induce with. 1 mM IPTG and 1 mM 5-ALA and let the expression go on for 48 hours at 25 C. I collect the cell pellet and lyse in a buffer composed of 50 mM tris HCl pH 7.4 with 150 mM NaCl, 20% glycerol, 0.4% triton x 100 or 1% CHAPS (I’ve tried both), 1 mM PMSF, 0.5 mM DTT, 10 mM imidazole, and an EDTA free protease inhibitor cocktail. I then centrifuge at 21000g for about an hour to collect the membrane debris. I load this onto the column using an Akta fplc with a flow rate of 0.5 mL per minute (25 mL column). My wash buffers are 50 mM tris HCl pH 7.4, 20% glycerol, 0.4% triton X 100 or 1% CHAPS, 250 mM NaCl. Buffer B consists of the same but with 250 mM imidazole and I use the Akta to wash the column and slowly increase the imidazole gradient up to 250 mM. Most of the protein comes out when flowing the lysate or during the 20 mM and imidazole wash. I have tried checking the pH of the buffers, pH of lysate, swapping detergents, changing salt concentration, my lysis protocol is 15 min total 15 seconds on 45 seconds off on a 1200W probe tip sonicator. I am honestly at my wits end and no one can tell me where it’s going wrong. I have this same protein with a 4x his tag (previously used by another group), a 6x his tag, and a 9x his tag and get the same result. The protein expresses well and we have sequenced the plasmids for each construct and they are correct

This purification using these conditions was done by another group previously. I emailed them asking for their protocols or advice and got a non answer (gotta love academics). We’ve run western blots and gels on the lysate and flow through and have confirmed the presence of the protein. UV vis also shows the correct soret peak for P450. I need some help or divine intervention here.

Edit: I am using the exact same plasmid used by another group to express and purify this protein. I received it directly from them and sequenced it. The tag is C terminal and I am tryin to purify using IMAC Ni-NTA resin.

So I recently started a postdoc and have been tasked with purifying a microsomal P450 enzyme. I did purification of his tagged heme proteins during my PhD and ever had any trouble. This P450 will not stick to the column no matter what I do.

EDIT: I’m purifying using a Ni-NTA resin and the protein is his tagged

Here is a brief explanation of my protocol and what I have tried. I grow the cells in TB media and induce with. 1 mM IPTG and 1 mM 5-ALA and let the expression go on for 48 hours at 25 C. I collect the cell pellet and lyse in a buffer composed of 50 mM tris HCl pH 7.4 with 150 mM NaCl, 20% glycerol, 0.4% triton x 100 or 1% CHAPS (I’ve tried both), 1 mM PMSF, 0.5 mM DTT, 10 mM imidazole, and an EDTA free protease inhibitor cocktail. I then centrifuge at 21000g for about an hour to collect the membrane debris. I load this onto the column using an Akta fplc with a flow rate of 0.5 mL per minute (25 mL column). My wash buffers are 50 mM tris HCl pH 7.4, 20% glycerol, 0.4% triton X 100 or 1% CHAPS, 250 mM NaCl. Buffer B consists of the same but with 250 mM imidazole and I use the Akta to wash the column and slowly increase the imidazole gradient up to 250 mM. Most of the protein comes out when flowing the lysate or during the 20 mM and imidazole wash. I have tried checking the pH of the buffers, pH of lysate, swapping detergents, changing salt concentration, my lysis protocol is 15 min total 15 seconds on 45 seconds off on a 1200W probe tip sonicator. I am honestly at my wits end and no one can tell me where it’s going wrong. I have this same protein with a 4x his tag (previously used by another group), a 6x his tag, and a 9x his tag and get the same result. The protein expresses well and we have sequenced the plasmids for each construct and they are correct

This purification using these conditions was done by another group previously. I emailed them asking for their protocols or advice and got a non answer (gotta love academics). We’ve run western blots and gels on the lysate and flow through and have confirmed the presence of the protein. UV vis also shows the correct soret peak for P450. I need some help or divine intervention here.