Soluble Intein Fusion Proteins And Methods For Purifying Biomolecules
AssigneeMerck Patent GmbH
InventorsMartin Zillmann, Joe Orlando
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Abstract
The present invention relates to fusion proteins comprising an N-intein polypeptide and an N-intein solubilization partner, and affinity chromatography matrices comprising such fusion proteins, as well as methods of using same.
Claims43
A fusion protein comprising an N-intein polypeptide and an N-intein solubilization partner joined by a peptide bond, wherein the N-intein solubilization partner has a molecular weight of less than about 15 kDa, an Aliphatic Index value less than about 60, and a Grand Average Hydropathy value less than −1, and enhances solubility of the N-intein polypeptide compared to the N-intein polypeptide expressed in the absence of the solubilization partner.
A method of affinity purifying a target molecule in a sample, the method comprising:
- a) providing a sample containing a first fusion protein comprising a C-intein polypeptide joined to a target molecule by a peptide bond;
- b) contacting the sample with an affinity chromatography matrix that comprises a second fusion protein, wherein the second fusion protein comprises an N-intein polypeptide joined by a peptide bond to an N-intein solubilization partner that promotes solubility of the N-intein polypeptide, under conditions in which the C-intein polypeptide in the first fusion protein selectively binds to the N-intein polypeptide in the second fusion protein to form an intein complex that is inactive;
- c) washing the affinity chromatography matrix containing the inactive intein complex to remove unbound contaminants;
- d) exposing the intein complex to conditions under which the intein complex is active and cleaves the target molecule from the C-intein polypeptide;
- and
- e) recovering the cleaved target molecule.
A method of screening for an intein complex that is suitable for use in affinity purification, the method comprising:
- a) contacting a first fusion protein that comprises a C-intein polypeptide joined to a target molecule by a peptide bond with a second fusion protein that comprises an N-intein polypeptide joined to an N-intein solubilization partner by a peptide bond, under conditions in which the C-intein polypeptide in the first fusion protein selectively binds to the N-intein polypeptide in the second fusion protein to form an intein complex;
- and
- determining whether the target molecule is cleaved from the C-intein polypeptide under conditions which support intein activity, wherein the presence of the cleaved target molecule is indicative of a catalytically-active intein complex.
Dependencies are inferred from the claim text.