Refolding Proteins: Difference between revisions
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==Introduction== | ==Introduction== | ||
Denaturing and then refolding proteins can be a good way to increase yield, because purification in denaturing conditions reduces nonspecific binding. You do, however, risk misfolding the protein. | |||
In other cases, recombinant or fusion proteins may be unfavorable to host bacteria, especially at the concentrations desired by researchers. As a result, these proteins may be misfolded and segregated into inclusion bodies. It may be necessary to purify the denatured protein from these inclusion bodies and then refold them manually. | |||
Please note that the below protocols do not involve purification from inclusion bodies. To do so, you can add lysozyme to reach 1 mM to the cell pellet, then vortex for 1 minute. | |||
==Resources== | ==Resources== | ||
*[[Knight:Purification_of_His-tagged_proteins/Denaturing_with_refolding]] - Qiagen Ni NTA spin columns | *[[Knight:Purification_of_His-tagged_proteins/Denaturing_with_refolding]] - Small scale (up to 0.5 to 1 mg protein, from 50mL culture) - Qiagen Ni NTA spin columns | ||
*[[Sauer:Purification_of_His-tagged_proteins/Denaturing_prep]] - Qiagen Ni NTA '''resin''' (goo). | *[[Sauer:Purification_of_His-tagged_proteins/Denaturing_prep]] - Large scale (1 L culture) - Qiagen Ni NTA '''resin''' (goo). | ||
*[http://refold.med.monash.edu.au/ REFOLD] - Repository of refolding protocols for specific protocols. | *[http://refold.med.monash.edu.au/ REFOLD] - Repository of refolding protocols for specific protocols. | ||
Latest revision as of 12:22, 14 August 2008
Introduction
Denaturing and then refolding proteins can be a good way to increase yield, because purification in denaturing conditions reduces nonspecific binding. You do, however, risk misfolding the protein.
In other cases, recombinant or fusion proteins may be unfavorable to host bacteria, especially at the concentrations desired by researchers. As a result, these proteins may be misfolded and segregated into inclusion bodies. It may be necessary to purify the denatured protein from these inclusion bodies and then refold them manually.
Please note that the below protocols do not involve purification from inclusion bodies. To do so, you can add lysozyme to reach 1 mM to the cell pellet, then vortex for 1 minute.
Resources
- Knight:Purification_of_His-tagged_proteins/Denaturing_with_refolding - Small scale (up to 0.5 to 1 mg protein, from 50mL culture) - Qiagen Ni NTA spin columns
- Sauer:Purification_of_His-tagged_proteins/Denaturing_prep - Large scale (1 L culture) - Qiagen Ni NTA resin (goo).
- REFOLD - Repository of refolding protocols for specific protocols.
See also
- IGEM:IMPERIAL/2007/CFS - Cell free systems for protein expression.
