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Protein refolding technics
I try to refold recomb Hiv1p24 protein from E-coli (his tagged) The native protein is membrane protein of inner core of HIV Analysing literature concerning protein refolding I find two main strategies/ First one - is step dialys against refolding buffer and other one diluting unfolded protein in refolding buffer. The third way concerning on column refolding I've tried but it was not sucessfull. What kind of two previous technics is more efficient from the term of refolding mechanism.
The other question is about composotion of refolding buffer. My protein hydrohobic protein with one internal disulfide bridge and one C-terminal cystein.
As I know L Arginine is important to prevent aggregation. Is it important to add cosolvents 1,5M urea or 1M Gua-Hcl?
What kind of ox\redox system is may be efficient GSH-GssG or TCEP or DTT or BME?
What role play glycerine, MgCl2, EDTA , CaCl2 ( targeted protein is not dependent on metal ions)?
What play and extent of importance of PEG, ionic strength(concentration of NaCl) and buffer system for refolding Tris or NaP
Is it very important to make pH of refolding buffer far from pI and in what side?
What about Chaps addition into RB?
What about temperature of Refolding ( in literature I've seen from 4, 15 to 25 C. )
I know about kits for refolding - Novagen (iFold) and Pierce. There are different kit of reagents. For example Novagen suggest N lauryl sarcosine for solubilization of inclusion bodies and Pirce recommend to solubilize IB in 6M Gua-Hcl / Is it difference important and to what extent?
Thank you in advance!
Re: Protein refolding technics
did you have any luck in refolding and how?
Re: Protein refolding technics
Wow - you've done quite a bit of work in screening different refolding conditions. Keep in mind that there's also a second step that can get in your way: the properly refolded protein may aggregate as you store or concentrate it. Therefore you may want to carefully optimize it's solubility behavior. Finding the 'solubility sweet spot' can be done with protein solubility screening kits such as the OptiSol protein solubility screening kit from Dilyx.
|protein , refolding , technics|
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