Method for purifying protein complexes

摘要

The present invention provides a method for the purification of complexes comprising a stress protein complexed to a peptide or peptide fragment, from a source mixture, typically a cell lysate. The improved method of the invention provides for protein complexes to be purified using ion exchange based methods, without the need to use chemicals such as chaotropes and ampholytes. The purified complexes can be used as the immunogenic determinant in vaccine compositions for the treatment or prevention of infectious diseases or cancerous conditions.

主权项

1. A method for purification of complexes formed between stress proteins of different stress protein families and an antigenic polypeptide or fragment thereof from a source mixture, wherein the polypeptide or fragment thereof is a tumor or pathogen polypeptide antigen, consisting of the steps of:
(i) providing the source mixture comprising target stress protein complexes which are to be purified from the source mixture wherein the target stress protein complexes are formed by proteins of different stress protein families complexed to the polypeptide or a fragment thereof, (ii) determining the isoelectric point (pI) of the target stress protein complexes; (iii) preparing a cell lysate from the source mixture comprising the target stress protein complexes; (iv) clarifying the cell lysate by subjecting the cell lysate to centrifugation and/or filtration to provide a clarified cell lysate; (v) transferring the clarified cell lysate of step (iv) to an ion exchange column wherein the transferred clarified cell lysate comprises all the stress protein complexes found within the cell lysate of step (iii); and (vi) subjecting the clarified cell lysate of step (v) to purification using an ion exchange solid phase, wherein during ion exchange the clarified cell lysate is buffered using a buffer to a pH within 2 units of the pI of the target stress protein complexes, and wherein a salt gradient provided by an elution buffer having a pH of from pH 4 to pH 9 is used to elute the target stress protein complexes comprising the stress proteins of the different stress protein families complexed to the polypeptide or a fragment thereof from the ion exchange solid phase, wherein the eluted target stress protein complexes comprise stress proteins from different stress protein families.