Methods and compositions for expressing functional class XIV myosin

Patent metadata

Field	Value
Publication number	US-9783584-B2
Application number	US-201615201588-A
Country	US
Kind code	B2
Filing date	Jul 4, 2016
Priority date	May 15, 2014
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

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 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 myosin light chain polypeptide, and wherein the encoded parasite co-chaperone polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 4 or a functional variant thereof that has an amino acid sequence with at least 75% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 12 or a functional fragment thereof that has an amino acid sequence with at least 75% sequence similarity to SEQ ID NO: 12.

2. The method of claim 1 , wherein the encoded parasite co-chaperone polypeptide functional variant comprises an amino acid sequence having at least 80% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 12.

3. The method of claim 1 , wherein the encoded parasite co-chaperone polypeptide functional variant comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 12.

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

5. The method of claim 4 , wherein at least one of:
(i) the amino acid sequence of the XIV heavy chain polypeptide functional variant has at least 80% similarity to the amino acid sequence of the wild-type class XIV heavy chain polypeptide or a functional fragment thereof;
(ii) the amino acid sequence of the XIV heavy chain polypeptide functional variant has at least 85% similarity to the amino acid sequence of the wild-type class XIV heavy chain polypeptide or a functional fragment thereof;
(iii) the amino acid sequence of the XIV heavy chain polypeptide functional variant has at least 90% similarity to the amino acid sequence of the wild-type class XIV heavy chain polypeptide or a functional fragment thereof;
(iv) the amino acid sequence of the myosin light chain polypeptide functional variant has at least 80% similarity to the wild-type myosin light chain polypeptide or a functional fragment thereof;
(v) the amino acid sequence of the myosin light chain polypeptide functional variant has at least 85% similarity to the wild-type myosin light chain polypeptide or a functional fragment thereof and
(vi) the amino acid sequence of the myosin light chain polypeptide functional variant has at least 90% similarity to the amino acid sequence of the wild-type myosin light chain polypeptide or a functional fragment thereof.

6. The method of claim 4 , wherein the wild-type class XIV heavy chain polypeptide has the amino acid sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 16.

7. The method of claim 4 , wherein the wild-type myosin light chain polypeptide has an amino acid sequence set forth herein as SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 49.

8. The method of claim 4 , wherein the class XIV heavy chain polypeptide functional variant has the amino acid sequence set forth as SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

9. 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, wherein at least one of the expression vectors additionally comprises at least one polynucleotide sequence that encodes a detectable label polypeptide, and wherein the functional class XIV myosin polypeptide comprises the expressed detectable label polypeptide.

10. The method of claim 9 , 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.

11. The method of claim 10 , wherein the heterologous expression system is a baculovirus/insect cell expression system.

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

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

14. A method of identifying a candidate compound to inhibit a parasite that expresses a class XIV myosin polypeptide, the method comprising:
(a) contacting a functional class XIV myosin polypeptide prepared as set forth in claim 1 , with a candidate compound under conditions suitable to determine an activity of the class XIV myosin polypeptide;
(b) determining the activity of the contacted prepared functional class XIV myosin polypeptide; and
(c) comparing the determined activity with a control activity determination, wherein a decrease in the determined activity in the contacted prepared functional class XIV myosin polypeptide compared to the control activity determination identifies the compound as a candidate compound to inhibit a parasite that expresses the functional class XIV myosin polypeptide.

15. The method of claim 14 , wherein the encoded parasite co-chaperone polypeptide functional variant comprises an amino acid sequence having at least 80% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 12.

16. The method of claim 14 , wherein the encoded parasite co-chaperone polypeptide functional variant comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 12.

17. The method of claim 14 , wherein the class XI

Assignees

• Univ Of Vermont And State Agricultural College

Inventors

• Trybus Kathleen M
• Ward Gary E

Classifications

• G01N2333/445

  Plasmodium · CPC title

• C07K14/4716

  Muscle proteins, e.g. myosin, actin · 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

• C12Q1/34

  involving hydrolase · CPC title

• C07K14/445 Primary

  Plasmodium · CPC title