CYP protein purification strategy

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Feb 2, 2019, 1:42:10 AM2/2/19

to Phyre

I have been trying for last two months using E.coli(BL21) cells, but all of them coming out in inclusiob bodies:( Any suugestions please.

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Feb 2, 2019, 2:24:45 AM2/2/19

to Phyre

Hi

Can you tell us more about culture conditions? Bellows are well known conditions to solve the problem.

1 Lower the temperature after induction, try 25 degree or 16 degree after induction and harvest after O/N culture.

2 If you use IPTG for induction try lower concentration such as 0.1 or 0.01 m M.

3 Attach solubility tags to your protein of interest such as GST, MBP, SUMO, GB1.

4 Coexpression of protein chaperone such as GroEL.

Those are the strategy I actually tried and all of them showed some effect on protein precipitation.

Good luck!

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Feb 2, 2019, 5:19:31 AM2/2/19

to Phyre

Dear author(高橋清大),

Thank you so much for your kind reply.I used following protocol:

1.16 degree after induction and harvest for 48 hours using 2YT medium.

2 IPTG at 0.4m M.

3 MBP tagged.

4 Expression of protein using chaperone groES-groEL.

I also used induction medium as well. The results are little improved using 2YT medium.

My proteins comes to the inclusion bodies.

TIA.

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Feb 2, 2019, 6:15:21 AM2/2/19

to Phyre

Thank you so much for your kind reply and suggestions. I will give you update after i follow these ones.

On Saturday, February 2, 2019 at 2:42:10 PM UTC+8, Nahiyan Salauddin wrote:

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