Heparin affinity tag and applications thereof

Patent metadata

Field	Value
Publication number	US-2016145597-A1
Application number	US-201414378230-A
Country	US
Kind code	A1
Filing date	Jan 21, 2014
Priority date	Jan 21, 2014
Publication date	May 26, 2016
Grant date	—

Abstract

In one aspect, affinity tags for recombinant protein purification are described herein which, in some embodiments, can mitigate or overcome disadvantages of prior affinity tag systems. In some embodiments, for example, affinity tags described herein permit efficient elution of desired recombinant proteins with simplified solution systems, such as alkali metal salt solutions. An affinity tag described herein comprises an amino acid sequence including a repeating amino acid unit of BXXXBXX, wherein B is an amino acid selected from the group consisting of histidine, lysine and arginine and X is an amino acid selected from the group consisting of amino acids other than histidine, lysine and arginine.

First claim

1 . An affinity tag for protein purification comprising:
an amino acid sequence including a repeating amino acid unit of BXXXBXX, wherein B is an amino acid selected from the group consisting of histidine, lysine and arginine and X is an amino acid selected from the group consisting of amino acids other than histidine, lysine and arginine.

2 . The affinity tag of claim 1 , wherein the affinity tag binds heparin.

3 . The affinity tag of claim 1 , wherein amino acid sequence comprises the sequence of SEQ ID NO:1.

4 . The affinity tag of claim 1 , wherein amino acid sequence comprises the sequence of SEQ ID NO:2.

5 . A recombinant protein composition comprising
a first amino acid sequence of a target protein and an affinity tag coupled to the first amino acid sequence, the affinity tag comprising an amino acid sequence for binding heparin.

6 . The recombinant protein composition of claim 5 further comprising a protease cleavage site between the target protein amino acid sequence and the affinity tag amino acid sequence.

7 . The recombinant protein composition of claim 6 , wherein the protease cleavage site is a serine protease cleavage site or cysteine protease cleavage site.

8 . The recombinant protein composition of claim 7 , wherein the serine protease is thrombin.

9 . The recombinant protein composition of claim 7 , wherein the cysteine protease is a tobacco etch virus (TEV) protease.

10 . The recombinant protein composition of claim 5 , wherein the affinity tag amino acid sequence comprises a repeating amino acid unit of BXXXBXX, wherein B is an amino acid selected from the group consisting of histidine, lysine and arginine and X is an amino acid selected from the group consisting of amino acids other than histidine, lysine and arginine.

11 . The recombinant protein composition of claim 10 , wherein the amino acid unit is repeated at least three times in the affinity tag amino acid sequence.

12 . The recombinant protein composition of claim 5 , wherein the affinity tag amino acid sequence comprises the sequence of SEQ ID NO:1.

13 . The recombinant protein composition of claim 1 , wherein the affinity tag amino acid sequence comprises the sequence of SEQ ID NO:2.

14 . A recombinant DNA molecule encoding for a protein comprising a first amino acid sequence of a target protein and an affinity tag amino acid sequence for binding heparin.

15 . The recombinant DNA molecule of claim 14 , wherein the protein further comprises a protease cleavage site between the target protein amino acid sequence and the affinity tag amino acid sequence.

16 . The recombinant DNA molecule of claim 14 , wherein the affinity tag amino acid sequence comprises a repeating amino acid unit of BXXXBXX, wherein B is an amino acid selected from the group consisting of histidine, lysine and arginine and X is an amino acid selected from the group consisting of amino acids other than histidine, lysine and arginine.

17 . The recombinant DNA molecule of claim 16 , wherein the amino acid unit is repeated at least three times in the affinity tag amino acid sequence.

18 . The recombinant DNA molecule of claim 14 comprising the nucleotide sequence of SEQ ID NO:3.

19 . The recombinant DNA molecule of claim 14 comprising the nucleotide sequence of SEQ ID NO:4.

20 . A method of purifying a recombinant protein composition comprising:
providing a mixture including the recombinant protein composition, the recombinant protein composition comprising a first amino acid sequence of a target protein and an affinity tag coupled to the first amino acid sequence, the affinity tag including an amino acid sequence for binding heparin; loading the mixture onto a separation column having a stationary phase comprising heparin; binding the recombinant protein to heparin of the stationary phase; and eluting the recombinant protein from the separation column.

21 - 52 . (canceled)

Assignees

• Univ Arkansas

Inventors

• Thallapuranam Suresh Kumar
• Jayanthi Srinivas
• Morris Jacqueline
• Brown Alicia
• Mcnabb David
• Henry Ralph

Classifications

• C12Y304/21043

  Classical-complement-pathway C3/C5 convertase (3.4.21.43) · CPC title

• C12N9/6424 Primary

  Serine endopeptidases (3.4.21) · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• B01J20/286

  Phases chemically bonded to a substrate, e.g. to silica or to polymers · CPC title