MSP nanopores and related methods

Patent metadata

Field	Value
Publication number	US-9540422-B2
Application number	US-201614993952-A
Country	US
Kind code	B2
Filing date	Jan 12, 2016
Priority date	Sep 22, 2008
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

Provided herein are Mycobacterium smegmatis porin nanopores, systems that comprise these nanopores, and methods of using and making these nanopores. Such nanopores may be wild-type MspA porins, mutant MspA porins, wild-type MspA paralog porins, wild-type MspA homolog porins, mutant MspA paralog porins, mutant MspA homolog porins, or single-chain Msp porins. Also provided are bacterial strains capable of inducible Msp porin expression.

First claim

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A Mycobacterium smegmatis porin A (MspA) comprising an MspA monomer, wherein the MspA monomer comprises a variant of the sequence in SEQ ID NO: 1, and wherein the variant comprises (i) amino acids with a neutral charge at positions 90 and 91 and (ii) an amino acid with a positive charge at one or more of amino acid positions 118, 134, and 139.

2. The MspA of claim 1 , wherein the variant comprises an amino acid with a positive charge at position 118.

3. The MspA of claim 1 , wherein the variant comprises an amino acid with a positive charge at positions 118, 134, and 139.

4. The MspA of claim 1 , wherein the variant comprises arginine at position 118.

5. The MspA of claim 1 , wherein the variant comprises lysine at position 139.

6. The MspA of claim 1 , wherein the variant comprises arginine at position 134.

7. The MspA of claim 1 , wherein the variant comprises arginine at position 118 and lysine at position 139.

8. The MspA of claim 3 , wherein the variant comprises arginine at position 118, arginine at position 134, and lysine at position 139.

9. The MspA of claim 1 , wherein the variant comprises asparagine or glutamine at position 90 and asparagine or glutamine at position 91.

10. The MspA of claim 1 , wherein the variant comprises asparagine at positions 90 and 91.

11. The MspA of claim 1 , wherein the variant comprises glutamine at positions 90 and 91.

12. The MspA of claim 1 , wherein the variant further comprises an amino acid substitution at position 88.

13. The MspA of claim 3 , wherein the variant further comprises an amino acid substitution at position 88.

14. The MspA of claim 9 , wherein the variant further comprises an amino acid substitution at position 88.

15. The MspA of claim 1 , wherein the variant further comprises an amino acid substitution at position 105.

16. The MspA of claim 1 , wherein the variant furthercomprises an amino acid substitution at position 108.

17. The MspA of claim 1 , wherein the variant further comprises an amino acid with a positive charge at position 126.

18. The MspA of claim 1 , wherein the variant further comprises aspartic acid at position 93.

19. The MspA of claim 3 , wherein the variant further comprises aspartic acid at position 93.

20. The MspA of claim 9 , wherein the variant comprises aspartic acid at position 93.

21. The MspA of claim 1 , wherein the variant comprises asparagine at positions 90 and 91, arginine at positions 118 and 134, and lysine at position 139, and further comprises asparagine at position 93.

22. The MspA of claim 1 , wherein the MspA further comprises a second MspA monomer, and wherein the MspA monomer is connected to the second MspA monomer by one or more amino acid linkers.

Assignees

• Univ Washington
• Uab Res Found

Inventors

• Gundlach Jens H
• Niederweis Michael
• Butler Thomas Z
• Pavlenok Mikhail
• Troll Mark A
• Sukumaran Suja

Classifications

• G01N33/6872

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C07K14/35 Primary

  from Mycobacteriaceae (F) · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title