SORTASE MOLECULES AND USES THEREOF

• US 20170226495A1
• Filed: 07/21/2015
• Published: 08/10/2017
• Est. Priority Date: 07/21/2014
• Status: Abandoned Application

• Current Assignee: Novartis Ag
• Original Assignee: Novartis Ag
• Inventors: Guimaraes, Carla
• Application Number:
  US15/327,816
• Publication Number:
  US 20170226495A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12Y 304/2207 Sortase A (3.4.22.70)

Abstract

This application provides mutant sortase molecules and methods of making and using them. In a first aspect, disclosed herein, are sortase molecules having one or a combination of mutations. In an embodiment, a sortase molecule is optimized for a parameter of enzyme performance, e.g., Ca++ dependency (or independency) or reaction rate.

Claims

1. A sortase molecule, or a purified or isolated preparation thereof, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94 (P94), Asp160 (D160), Asp165 (D165), Lys190 (K190) and Lys196 (K196); and

a mutation selected from Glu105 (E105) and Glu108 (E108); and

having at least 90% homology with SEQ ID NO;

3.

2. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94 (P94), Asp160 (D160), Asp165 (D165), Lys190 (K190) and Lys196 (K196); and

a mutation selected from Glu105 (E105) and Glu108 (E108); and

otherwise differing from SEQ ID NO;

3 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

3. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94 (P94), Asp160 (D160), Asp165 (D165), Lys190 (K190) and Lys196 (K196); and

a mutation selected from Glu105 (E105) and Glu108 (E108).

4. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94Arg (P94R), Asp160Asn (D160N), Asp165Ala (D165A), Lys190Glu (K190E) and Lys196Thr (K196T); and

a mutation selected from Glu105Lys (E105K) and Glu108Gln (E108Q).

5. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94Arg (P94R), Asp160Asn (D160N), Asp165Ala (D165A), Lys190Glu (K190E) and Lys196Thr (K196T); and

a mutation selected from Glu105Lys (E105K) and Glu108Gln (E108Q); and

having at least 90% homology with SEQ ID NO;

3.

6. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising;

a mutation selected from Pro94Arg (P94R), Asp160Asn (D160N), Asp165Ala (D165A), Lys190Glu (K190E) and Lys196Thr (K196T); and

a mutation selected from Glu105Lys (E105K) and Glu108Gln (E108Q); and

otherwise differing from SEQ ID NO;

3 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

7. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising the following mutations;

Pro94 (P94), Glu105 (E105), Glu108 (E108), Asp160 (D160), Asp165 (D165), Lys190 (K190) and Lys196 (K196); and

having at least 90% homology with SEQ ID NO;

3.

8. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising the following mutations;

Pro94 (P94), Glu105 (E105), Glu108 (E108), Asp160 (D160), Asp165 (D165), Lys190 (K190) and Lys196 (K196) and otherwise differing from SEQ ID NO;

3 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

9. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

3, comprising the following mutations;

Pro94 (P94), Glu105 (E105), Glu108 (E108), Asp160 (D160), Asp165 (D165), Lys190 (K190), and Lys196 (K196).

10. The sortase molecule of claim 1, which comprises the amino acid sequence of SEQ ID NO:

5.

11. A nucleic acid encoding the sortase molecule of claim 1.

12. A vector comprising the nucleic acid of claim 11.

13. A cell comprising the nucleic acid of claim 11.

14. A method of making a sortase molecule, comprising, providing a cell comprising a nucleic acid or vector that comprises sequence that encodes the sortase molecule of claim 1, and recovering the sortase molecule from the cell or secreted by the cell.

15. A method of coupling a first moiety to a second moiety, comprising:

a) providing the first moiety coupled to a sortase acceptor motif and the second moiety coupled to a sortase recognition motif;

b) contacting the first moiety coupled to a sortase acceptor motif with;

(i) a sortase molecule and the second moiety coupled to a sortase recognition motif;

or

(ii) a complex comprising the second moiety coupled to a cleaved sortase recognition motif and a sortase molecule;

under conditions sufficient to allow transfer of a second moiety coupled to a cleaved sortase recognition motif to the sortase acceptor motif coupled to the first moiety,

thereby coupling a first moiety to a second moiety,

provided that, the sortase molecule is the sortase molecule of claim 1.

16. A method of providing a cell having a moiety attached thereto, comprising

a) providing a sortase acceptor motif coupled to a first moiety disposed in or on a precursor cell;

b) contacting the precursor cell with

(i) a sortase molecule and a second moiety coupled to a sortase recognition motif;

or

(ii) a complex comprising the second moiety coupled to a cleaved sortase recognition motif and a sortase molecule,

under conditions sufficient to allow transfer of a second moiety coupled to a cleaved sortase recognition motif to the sortase acceptor motif coupled to the first moiety,

provided that, the sortase molecule is the sortase molecule of claim 1,

thereby providing cell having a moiety attached thereto.

17. A method of providing a purified preparation of a first moiety coupled to a second moiety, comprising:

providing the first moiety coupled to the second moiety, and

separating the first moiety coupled to the second moiety from a sortase molecule,

thereby providing a purified preparation of a first moiety coupled to a second moiety,

wherein the sortase molecule is the sortase molecule of claim 1.

18. (canceled)