Supramolecular filamentous assemblies for protein purification
The present invention provide novel immunofiber compositions for protein or peptide purification and simple and cost-efficient methods and systems using these compositions. In some embodiments, the immunofibers comprise a customized Z-33 peptide derived from Staphylococcus aureus Protein A which is used to construct immuno-amphiphile molecules that assemble into immunofibers in aqueous solution with bioactive epitopes on the surface and have peptide or protein binding ability.
1. An immunofiber composition comprising one or more immuno-amphiphiles, wherein said immuno-amphiphiles comprise an antibody binding peptide conjugated to a hydrocarbon chain; and wherein the antibody binding peptide comprises a hydrophilic amino acid sequence of the Z33 peptide of Protein A of Staphylococcus aureus or an antibody-binding fragment of Z33.
2. The immunofiber composition of claim 1 , wherein the immuno-amphiphile has an α-helical conformation when in an aqueous solution at physiological pH.
3. The immunofiber composition of claim 1 , wherein the antibody binding peptide comprises the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1) or an antibody-binding fragment of SEQ ID NO: 1.
4. The immunofiber composition of claim 1 , wherein the antibody binding peptide has a hydrophilic amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.
5. The immunofiber composition of claim 1 , wherein the hydrocarbon chain is between 8 and 22 carbons in length and is either linear or branched.
6. The immunofiber composition of claim 5 , wherein the hydrocarbon chain is linear.
7. The immunofiber composition of claim 6 , wherein the hydrocarbon chain is between 8 and 12 carbons in length.
8. An immunofiber composition comprising an immunofiber binding molecule, wherein:
said immunofiber binding molecule comprises an antibody binding peptide conjugated at its N-terminus to a spacer peptide and said spacer peptide is conjugated at its N-terminus to a hydrocarbon chain;
said antibody binding peptide has a hydrophilic amino acid sequence of the Z33 peptide of Protein A of Staphylococcus aureus , an antibody-binding fragment of Z33, or an antibody-binding derivative of Z33; and
said spacer peptide comprises the generic amino acid sequence of XXYYZZ, wherein XX is two amino acids having a small hydrophobic side chain and can be the same or different amino acid, YY is two amino acids having a positively charged side chain and can be the same or different amino acid, and ZZ is two amino acids having a small neutral side chain and can be the same or different amino acid.
9. The immunofiber composition of claim 8 , wherein the antibody binding peptide has the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1), an antibody-binding fragment of SEQ ID NO: 1, or an antibody-binding derivative of SEQ ID NO: 1.
10. The immunofiber composition of claim 8 , wherein the antibody binding peptide has a hydrophilic amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.
11. The immunofiber composition of claim 8 , wherein the hydrocarbon chain is 8 carbons in length.
12. The immunofiber composition of claim 8 , wherein the amino acids having a small hydrophobic side chain are selected from the group consisting of: Ala, Val, Ile, and Leu.
13. The immunofiber composition of claim 8 , wherein the amino acids with a positively charged side chain are selected from the group consisting of: Arg, His and Lys.
14. The immunofiber composition of claim 8 , wherein the amino acids with a small neutral side chain are selected from the group consisting of: Gly and Pro.
15. A method for purifying antibodies or Fc containing peptides or proteins, the method comprising:
a) dissolving immuno-amphiphiles in an aqueous solution having a physiological pH to provide an immuno-amphiphile solution; and aging said immuno-amphiphile solution overnight to provide an immunofiber solution comprising immunofibers, wherein said immuno-amphiphiles comprise an antibody binding peptide conjugated to a hydrocarbon chain;
b) mixing a sample containing antibodies or Fc containing peptides or proteins with the immunofiber solution, and allowing the immunofibers to bind the Fc portion of the antibodies or Fc containing peptides or proteins and form an immunofiber-antibody complex or immunofiber-Fc containing peptide or protein complex in solution;
c) separating the immunofiber-antibody complex or immunofiber-Fc containing peptide or protein complex from the solution by adding salt and centrifugation;
d) dissociating the immunofibers from the antibodies or Fc containing peptides or proteins and collecting the unbound antibodies or Fc containing peptides or proteins.
16. The method of claim 15 , further comprising:
e) separating the immunofibers from the antibodies or Fc containing peptides or proteins by lowering the pH to elution condition and filtration, microfiltration, or ultrafiltration.
17. The method of claim 15 , wherein the immunoglobulins or Fc containing peptides or proteins are further purified using polishing steps.
18. The method of claim 16 , wherein the immunoglobulins or Fc containing peptides or proteins are further purified using polishing steps.