Precipitable peptides

Patent metadata

Field	Value
Publication number	US-9550805-B2
Application number	US-201314052367-A
Country	US
Kind code	B2
Filing date	Oct 11, 2013
Priority date	Apr 13, 2011
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

The invention is directed to a Ca2+ precipitable polypeptide tags and cassettes useful for purification of molecules from heterogeneous samples. The invention also relates to methods for bioseparation of molecules comprising Ca2+ precipitable tags and cassettes.

First claim

What is claimed is:

1. A precipitable beta roll cassette (PBRC) comprising at least two beta roll tags (PBRTs) wherein the at least two PBRTs each comprise the amino acid sequence of SEQ ID NO: 1, wherein the PBRC precipitates in response to binding of calcium by the at least two PBRTs.

2. A precipitable beta roll cassette (PBRC) comprising at least two beta roll tags (PBRTs) wherein the at least two PBRTs are independently any of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or
(b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337;
wherein the PBRC precipitates in response to binding of calcium by the at least two PBRTs.

3. A precipitable beta roll cassette (PBRC) comprising at least two beta roll tags (PBRTs) wherein the at least two PBRTs are independently any of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or
(b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337; or
(c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein,
(i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and
(ii) the X at position 3 is an amino acid selected from the group consisting of alanine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and
(iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and
(iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and
(v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine,
(vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and
(vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and
(viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine;
wherein the PBRC precipitates in response to binding of calcium by the at least two PBRTs.

4. The PBRC of any of claims 1 - 3 further comprising a capping sequence.

5. The PBRC of any of claims 1 - 3 further comprising a stabilizing polypeptide.

6. A PBRC linked purification moiety comprising the PBRC of any of claims 1 - 3 .

7. The PBRC linked purification moiety of claim 6 wherein the PBRC is linked to the purification moiety by a peptide bond.

8. The PBRC linked purification moiety of claim 6 wherein the PBRC is linked to the purification moiety by a chemical bond that is not a peptide bond.

9. The PBRC of any of claims 1 - 3 further comprising a cleavage site located N-terminally or C-terminally to one or more of the at least two PBRTs.

10. The PBRC of claim 9 , wherein the cleavage site is selected from the group comprising an intein cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, an enterokinase cleavage site, or a signal peptidase cleavage site.

11. A polypeptide comprising the PBRC of any of claims 1 - 3 and a purification moiety.

Assignees

• Univ Columbia

Inventors

• Banta Scott
• Shur Oren

Classifications

• C07K2319/92

  containing an intein ("protein splicing")domain · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C07K14/245

  Escherichia (G) · CPC title

• C07K1/303 Primary

  by salting out · CPC title

• C07K1/30 Primary

  by precipitation · CPC title