Process for renaturation of polypeptides
AssigneeBiogenomics Limited
InventorsArchana Rajesh KRISHNAN, Sanjay Madhukar SONAR, Nikhil Sudhir GHADE
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Abstract
The invention relates to method of refolding of proteins from a solution containing the protein in predominantly misfolded, aggregated form. The method involves denaturation and reduction of the protein of interest. The denatured and reduced preparation is subjected to removal of reducing agent in denaturing condition and at low pH to prevent the misfolding of the protein. The protein preparation is subjected to refolding followed by removal of the refolding buffer components.
Claims20
A method for obtaining, a preparation of protein where substantial part of the protein is in a refolded form from a preparation comprising a mixture of protein in an aggregated, unfolded and misfolded form, the method comprising the sequence of steps of:
- (i) adding a denaturing concentration of chaotropic agent to the first preparation comprising a mixture of protein in an aggregated, unfolded and misfolded form, to obtain a second preparation comprising the mixture of protein in a denatured form;
- (ii) adding a reducing agent to the second preparation comprising the mixture of protein in a denatured form, incubating the said mixture for sufficient time at appropriate temperature to obtain a third preparation comprising the protein in a substantially monomeric form;
- (iii) Lowering the pH of the said third preparation and removing the reducing agent in the presence of denaturing concentration of chaotropic agent while maintaining low pH to obtain a fourth preparation comprising the protein in a substantially monomeric form;
- (iv) diluting the said fourth preparation with appropriate volume of refolding buffer of alkaline pH comprising a refolding agent and non denaturing concentration of chaotropic agent to obtain a fifth preparation where substantial part of the protein is refolded, and
- (v) Separating the chaotropic agent and components of refolding buffer from said fifth preparation to obtain a desalted form of refolded protein preparation.
A method for obtaining, a preparation of protein which is devoid of any reducing agent and where substantial part of the protein is in an unfolded, monomeric form from a preparation comprising a mixture of protein in an aggregated, unfolded and misfolded form, the method comprising the sequence of steps of:
- (i) adding a denaturing concentration of chaotropic agent to the first preparation comprising a mixture of protein in an aggregated, unfolded and misfolded form, to obtain a second preparation comprising the mixture of protein in a denatured form;
- (ii) adding a reducing agent to the second preparation comprising the mixture of protein in a denatured form, incubating the said mixture for sufficient time at appropriate temperature to obtain a third preparation comprising the protein in a substantially monomeric form;
- (iii) Lowering the pH of the said third preparation and removing the reducing agent in the presence of denaturing concentration of chaotropic agent while maintaining low pH to obtain a fourth preparation comprising the protein in a substantially monomeric form, wherein the protein solution obtained is substantially devoid of any reducing agent.
Dependencies are inferred from the claim text.