Methods and compositions for expressing functional class XIV myosin

Patent metadata

Field	Value
Publication number	US-9409962-B2
Application number	US-201514712466-A
Country	US
Kind code	B2
Filing date	May 14, 2015
Priority date	May 15, 2014
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

The invention, in part, includes methods and compounds useful to prepare and functional class XIV myosin. Functional class XIV myosin prepared using methods of the invention may be useful to screen for and identify compounds that inhibit and treat parasitic infections and contamination.

First claim

What is claimed is:

1. A method of producing a functional class XIV myosin polypeptide, the method comprising,
co-expressing three or more polynucleotides in a heterologous expression-system cell, wherein the three or more polynucleotides comprise a class XIV heavy chain polypeptide-encoding polynucleotide, a first myosin light chain polypeptide-encoding polynucleotide, and a parasite co-chaperone polypeptide-encoding polynucleotide, wherein the three or more polynucleotides are co-expressed in the cell under conditions suitable to produce a functional class XIV myosin polypeptide comprising the class XIV heavy chain polypeptide and the first myosin light chain polypeptide.

2. The method of claim 1 , wherein the class XIV heavy chain polypeptide-encoding polynucleotide, the parasite co-chaperone polypeptide-encoding polynucleotide, and the first myosin light chain polypeptide-encoding polynucleotide are each independently selected from a Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria , or Cryptosporidium class XIV heavy chain polypeptide-encoding polynucleotide; a parasite co-chaperone polypeptide-encoding polynucleotide; and a myosin light chain polypeptide-encoding polynucleotide, respectively; and wherein the encoded class XIV heavy chain polypeptide comprises an amino acid sequence having at least 75% sequence similarity to a wild-type class XIV heavy chain polypeptide or a functional fragment thereof; the encoded first myosin light chain polypeptide comprises an amino acid sequence having at least 75% sequence similarity to a wild-type myosin light chain polypeptide or a functional fragment thereof; and the encoded parasite co-chaperone polypeptide comprises an amino acid sequence having at least 75% sequence similarity to a wild-type parasite co-chaperone polypeptide or a functional fragment thereof.

3. The method of claim 1 , wherein co-expressing comprises co-infecting the heterologous expression-system cell with one or more expression vectors each comprising one or more of the three or more polynucleotides.

4. The method of claim 3 , wherein the one or more expression vectors is a viral expression vector.

5. The method of claim 3 , wherein co-infecting the heterologous expression-system cell comprises co-infecting with one or more expression vectors comprising one or more of the class XIV heavy chain polypeptide-encoding polynucleotide operably linked to an independently selected promoter, the first myosin light chain polypeptide-encoding polynucleotide operably linked to an independently selected promoter, and the parasite co-chaperone polypeptide-encoding polynucleotide operably linked to an independently selected promoter.

6. The method of claim 3 , wherein co-infecting the heterologous expression-system cell comprises co-infecting with a first expression vector comprising the class XIV heavy chain polypeptide-encoding polynucleotide operably linked to an independently selected promoter, a second expression vector comprising the first myosin light chain polypeptide-encoding polynucleotide operably linked to an independently selected promoter, and a third expression vector comprising the parasite co-chaperone polypeptide-encoding polynucleotide operably linked to an independently selected promoter.

7. The method of claim 3 , wherein the one or more of the expression vectors additionally comprises one or more polynucleotides that encode: a myosin light chain-1 (MLC1) polypeptide having at least 75% similarity to the sequence of a wild-type MLC1 polypeptide or a functional fragment thereof; a tail domain interacting protein (MTIP) polypeptide having at least 75% similarity to the sequence of a wild-type MTIP polypeptide or a functional fragment thereof; an essential light chain-1 (ELC1) polypeptide having at least 75% similarity to the sequence of a wild-type ELC1 polypeptide or a functional fragment thereof; a calmodulin polypeptide having at least 75% similarity to the sequence of a wild-type calmodulin) polypeptide or a functional fragment thereof; or a glideosome associated protein-45 (GAP45) polypeptide having at least 75% similarity to the sequence of a wild-type GAP45 polypeptide or a functional fragment thereof.

8. The method of claim 3 , wherein one or more of the expression vectors additionally comprise at least one polynucleotide sequence that encodes a detectable label, and wherein the functional class XIV myosin polypeptide comprises the expressed detectable label polypeptide.

9. The method of claim 1 , wherein the parasite co-chaperone polypeptide-encoding polynucleotide sequence comprises a UNC-45/Cro1/She4p (UCS) chaperone polynucleotide sequence encoding a parasite co-chaperone polypeptide having at least 75% similarity to the sequence of a wild-type UNC-45/Cro1/She4p (UCS) polypeptide or a functional fragment thereof, respectively.

10. The method of claim 1 , wherein the encoded parasite co-chaperone polypeptide comprises an amino acid sequence having at least 75% similarity to the sequence of a wild-type toxoplasma gondii UCS-45 homolog or a functional fragment thereof; or a wild-type Toxoplasma UNC (TgUNC) polypeptide or a functional fragment thereof.

11. The method of claim 1 , wherein the parasite co-chaperone polypeptide comprises an amino acid sequence having at least 75% similarity to: the sequence of a wild-type Plasmodium falciparum UCS-45 homolog or a functional fragment thereof; or a wild-type Plasmodium falciparum UNC (PfUNC) polypeptide, or a functional fragment thereof.

12. The method of claim 11 , wherein the PfUNC functional fragment is a truncated PfUNC polypeptide.

13. The method of claim 1 , wherein the heterologous expression system is a baculovirus/insect cell expression system.

14. The method of claim 1 , wherein the first myosin light chain polypeptide is a regulatory light chain (MLC1) polypeptide comprising an amino acid sequence having at least 75% similarity to: a wild-type Toxoplasma gondii regulatory light chain polypeptide sequence or a functional fragment thereof; or a wild-type Plasmodium falciparum regulatory light chain polypeptide sequence or a functional fragment thereof.

15. The method of claim 1 , further comprising isolating the expressed functional class XIV myosin polypeptide.

16. The method of claim 1 , further comprising assaying the function of the expressed functional class XIV myosin polypeptide.

17. The method of claim 1 , further comprising co-infecting the heterologous expression-system cell with an expression vector comprising a second myosin light chain encoding polynucleotide; and
co-expressing the class XIV heavy chain polynucleotide, the first and second myosin light chain polynucleotides, and the parasite co-chaperone polynucleotide under conditions suitable to produce a functional class XIV myosin polypeptide comprising the class XIV heavy chain polypeptide and the first and second myosin light chain polypeptides.

18. A functional class XIV myosin polypeptide prepared by the method of claim 8 .

19. A method of determining an activity of a class XIV myosin polypeptide, the method comprising,
(a) preparing a functional class XIV myosin polypeptide with a method comprising co-expressing three or more polynucleotides in an expression-system cell, wherein the three or more polynucleotides comprise a class XIV heavy chain polypeptide-encoding polynucleotide, a first myosin light chain polypeptide-encoding polynucleotide, and a parasite co-chaperone polypeptide-encoding polynucleotide, wherein the three or more polynucleotides are co-expressed in the cell under

Assignees

• Univ Of Vermont And State Agricultural College

Inventors

• Trybus Kathleen M
• Ward Gary E

Classifications

• G01N2333/44

  from protozoa · CPC title

• G01N33/6887

  from muscle, cartilage or connective tissue · CPC title

• C12Q1/025

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title

• C07K14/45

  Toxoplasma · CPC title

• C07K14/44

  from protozoa · CPC title