Inclusion body purification & protein refolding

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Fornita
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Inclusion body purification & protein refolding

Post by Fornita » Fri Jun 17, 2016 3:51 am

Recombinant protein expression in bacteria often results in the formation of both inactive and insoluble protein that accumulates as intracellularprotein aggregates called inclusion bodies. It has beenshown that 70-80% of recombinant proteins expressed in E.coli are as inclusion bodies. This is probably due to the independence of the protein type in bacterial systems. In cases of expression of eukaryotic proteins, which usually contain cysteines that are prone to form disulfide bonds in the nativestate, the bacterial system maynot support the appropriate pairing of disulfide bonds in the newly-produced protein thus leads to the presence of insoluble protein pellets .
Inclusion bodies are not restricted to E.coli, they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E.coli cell wall and outer membranecomponents.

Here is what we do at Profacgen to obtain native and soluble protein form. First of all, the insoluble protein pellets must be separated from other cellular components by homogenization, washing and centrifugation; which is then followed by the refolding of protein by solubilization in denaturants, such as guanidine hydrochloride or urea. Besides, certain reducing reagents are added to reduce the polypeptide cysteines to break existing disulfide bonds to obtain monomeric peptide chains.

Generally speaking, we apply selective extraction with detergents and low concentrations of urea or guanidine chloride to solublize the protein pellets. These basic steps can dissolve about 60% of the pellet protein. The challenge, therefore, is not to purify the recombinantly-derived protein, but to solubilize it and then fold it into native and biologically active protein.

Profacgen possesses expertise in recovery of correctly folded protein, which requires laborious and expensive processing of inclusion bodies by conventional methods. We guarantee the structural integrity and native conformation of your target protein.
For more:http://www.profacgen.com/inclusion-body-purification-protein-refolding.htm
Andrea Fortina
Genius only means hard-working all one's life.
CreativeBiomart, Recombinant protein expert

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