2 replies to this topic

[mgn67]

Hi,

I’m trying to purify a his-tagged protein from baculovirus-infected insect cells.

First time, I just pelleted my cells, washed them with PBS/10% glycerol, pelleted again and lysed with 1M Nacl, 20mM Tris pH 7.5, 10% glycerol, 0.01% NP40 and PIC (my protein is tightly bound to DNA…). This first time, everything just worked fine, amazing expression, and everything was soluble !

Doing it again, it just didn’t work anymore… My protein went from entirely soluble to completely insoluble… I used the same conditions, the same buffer, so I don’t get why it’s not working anymore… I’ve been now trying for 3 months so solubilize it and it’s just impossible… I’ve screened different lysis conditions (urea, higher salt, different detergents, reducing agents, dounce homogenizer, different sonication time, fresh or frozen pellets…).

Do any of you have an idea on what I could try to solubilize this protein ?

Thanks a lot !
Morgan

Edit : I just want to add that I want to keep my protein native, my aim being to crystalize it... So, refolding could be considered but not as a first choice.

Edited by mgn67, 16 May 2011 - 03:10 AM.

[protolder]

mgn67, on 16 May 2011 - 01:29 AM, said:

Hi,

I’m trying to purify a his-tagged protein from baculovirus-infected insect cells.

First time, I just pelleted my cells, washed them with PBS/10% glycerol, pelleted again and lysed with 1M Nacl, 20mM Tris pH 7.5, 10% glycerol, 0.01% NP40 and PIC (my protein is tightly bound to DNA…). This first time, everything just worked fine, amazing expression, and everything was soluble !

Doing it again, it just didn’t work anymore… My protein went from entirely soluble to completely insoluble… I used the same conditions, the same buffer, so I don’t get why it’s not working anymore… I’ve been now trying for 3 months so solubilize it and it’s just impossible… I’ve screened different lysis conditions (urea, higher salt, different detergents, reducing agents, dounce homogenizer, different sonication time, fresh or frozen pellets…).

Do any of you have an idea on what I could try to solubilize this protein ?

Thanks a lot !
Morgan

Edit : I just want to add that I want to keep my protein native, my aim being to crystalize it... So, refolding could be considered but not as a first choice.

Hola, if you keep the same relation buffer /number of cells it´s extrange the different solubility. Second the virus passage and the titer are the same? so MOI is the same?. If I were you I will increase the volume of buffer to lyse the cells (it doesn´t affects to Ni column), and vary pH a bit more far  of isoelectric point of the protein. I would infect with less virus units, and harvest the infected cells a day before, if your infection time pass of 72 hours. Buena suerte

[mgn67]

Thanks for answering,

I indeed kept the same amount for lysis buffer (6 mL for a 250mL culture pellet) which was too little according to the technician of the baculovirus service. It waas though in this condition that my protein was soluble... anyway, she told me to use 80 to 100mL per liter of culture. It didn't change anything...

Then for the MOI, it's always the same (it's a service here in my institute which grows the cells and infects them) : 1 pfu, 48 hours of culture.

Changing the pH of my lysis buffer could be a solution, I will try this asap !

Thanks,
Morgan.