Precipitable peptides

Patent metadata

Field	Value
Publication number	US-10358461-B2
Application number	US-201615377243-A
Country	US
Kind code	B2
Filing date	Dec 13, 2016
Priority date	Apr 13, 2011
Publication date	Jul 23, 2019
Grant date	Jul 23, 2019

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 method for purifying a precipitable beta roll cassette (PBRC) linked purification moiety, the method comprising:
(a) expressing the PBRC linked purification moiety in an expression system,
(b) collecting the PBRC linked purification moiety in a first medium,
(c) adding Ca2+ to the first medium so as to induce precipitation of PBRC linked purification moiety, and
(d) removing unprecipitated material from the medium from the precipitated PBRC linked purification moiety;
wherein the PBRC comprises at least two beta roll tags (PBRTs), the at least two PBRTs being 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.

2. The method for purifying a PBRC linked purification moiety according to claim 1 , the method further comprising:
(e) resuspending the PBRC linked purification moiety in a second medium having a lower than the free Ca2+ concentration than the free Ca2+ concentration obtained after step (c).

3. The method of claim 2 wherein a calcium chelator is added to the second medium of step (e).

4. The method of claim 2 , wherein steps (c) to (e) are repeated one or more times.

5. The method of claim 1 , further comprising a step of removing precipitated material between step (b) and step (c).

6. The method of claim 1 , wherein the PBRC comprises a cleavage site between the PBRC and the purification moiety.

7. The method of claim 1 , further comprising steps of:
(i) cleaving the PBRC linked purification moiety so as to separate the purification moiety from the PBRC,
(ii) adding Ca2+ to the medium so as to induce precipitation of the PBRC, and
(iii) isolating the unprecipitated purification moiety.

8. The method of claim 6 , wherein the cleavage site is selected from the group consisting of an intein cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, an enterokinase cleavage site, or a signal peptidase cleavage site.

9. The method of claim 1 , wherein the at least two PBRTs each comprise the amino acid sequence of SEQ ID NO: 1.

10. The method of claim 1 , wherein the PBRC comprises a capping sequence.

11. The method of claim 1 , wherein the PBRC comprises a stabilizing peptide.

12. The method of claim 6 , wherein the cleavage site is located N-terminally or C-terminally to the PBRC.

13. A method for purifying a precipitable beta roll cassette (PBRC) linked purification moiety, the method comprising:
(a) expressing the PBRC linked purification moiety in an expression system,
(b) collecting the PBRC linked purification moiety in a first medium,
(c) adding Ca2+ to the first medium so as to induce precipitation of PBRC linked purification moiety, and
(d) removing unprecipitated material from the medium from the precipitated PBRC linked purification moiety;
wherein the PBRC comprises at least two beta roll tags (PBRTs), wherein the at least two PBRTs are independently any of:
a polypeptide comprising the amino acid sequence GXXXXXXXX (SEQ ID NO: 1343), 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, and
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.

Assignees

• Univ Columbia

Inventors

• Banta Scott
• Shur Oren

Classifications

• C07K2319/50

  containing protease site · CPC title

• C07K2319/20

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

• C07K1/30

  by precipitation · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/245

  Escherichia (G) · CPC title