Methods and compositions for rapidly replacing cardiac myosin binding protein-C in sarcomeres
View Patent ↗Methods and compositions for rapidly replacing cMyBP-C in sarcomeres featuring the creation of Spy-C mice, which are mice genetically engineered to express cMyBP-C with a protease recognition site and SpyTag peptide introduced into the cMyBP-C gene. In permeabilized myocytes from the Spy-C mice, the cMyBP-C protein can be cleaved at the protease recognition site, and the N-Terminus of cMyBP-C can be removed while the C-terminus remains anchored to the thick filament. A new peptide featuring the SpyCatcher sequence can be covalently bonded to the remaining portion of cMyBP-C, thereby creating a modified cMyBP-C protein. The methods and compositions of the present invention allow for the reconstitution of full-length cMyBP-C at the precise position of native cMyBP-C in the sarcomere and allow for a variety of modifications to be introduced to cMyBP-C in situ.
1. A recombinant protein comprising cMyBP-C with an internal insertion therein, the internal insertion comprises at least a SpyTag peptide adjacent to a protease recognition site, wherein the internal insertion is within or between C7 and C8 domains of cMyBP-C.
2. A recombinant protein comprising cMyBP-C with an internal insertion therein, the internal insertion comprises at least a SpyTag peptide adjacent to a protease recognition site, wherein the internal insertion is in between residues VQEILQR (SEQ ID NO: 14) in domain C7 and residues PRLQLPRH (SEQ ID NO: 15) of domain C8 of cMyBP-C.
3. A recombinant protein comprising cMyBP-C with an internal insertion therein, wherein the recombinant cMyBP-C peptide is according to SEQ ID NO: 13.
4. A method of producing a recombinant cMyBP-C protein, said method comprising:
a. introducing into a genome of a host a cassette for expressing a first recombinant cMyBP-C protein, wherein the first recombinant protein is according to the recombinant protein of claim 1 , wherein the protease recognition site is adjacent to and 5′ to the SpyTag peptide;
b. isolating myocytes from the host;
c. introducing to the myocytes a protease that cleaves the first cMyBP-C protein at the protease recognition site to remove its N-terminus and expose the SpyTag peptide;
d. introducing a recombinant cMyBP-C N-terminus, the recombinant cMyBP-C N-terminus being at least a portion of cMyBP-C with a SpyCatcher peptide at its C-terminus;
wherein the SpyCatcher peptide of the cMyBP-C N-terminus binds to the SpyTag peptide of the first recombinant cMyBP-C to produce a second recombinant cMyBP-C protein.
5. A motility assay platform comprising: a coverslip with a thick filament disposed thereon, wherein the thick filament comprises a recombinant cMyBP-C protein according to claim 1 .
6. A method of producing a recombinant cMyBP-C protein, said method comprising:
a. introducing into a genome of a host a cassette for expressing a first recombinant cMyBP-C protein, wherein the first recombinant protein is according to the recombinant protein of claim 2 , wherein the protease recognition site is adjacent to and 5′ to the SpyTag peptide;
b. isolating myocytes from the host;
c. introducing to the myocytes a protease that cleaves the first cMyBP-C protein at the protease recognition site to remove its N-terminus and expose the SpyTag peptide;
d. introducing a recombinant cMyBP-C N-terminus, the recombinant cMyBP-C N-terminus being at least a portion of cMyBP-C with a SpyCatcher peptide at its C-terminus;
wherein the SpyCatcher peptide of the cMyBP-C N-terminus binds to the SpyTag peptide of the first recombinant cMyBP-C to produce a second recombinant cMyBP-C protein.
7. A motility assay platform comprising: a coverslip with a thick filament disposed thereon, wherein the thick filament comprises a recombinant cMyBP-C protein according to claim 2 .
8. A method of producing a recombinant cMyBP-C protein, said method comprising:
a. introducing into a genome of a host a cassette for expressing a first recombinant cMyBP-C protein, the first recombinant cMyBP-C protein being a cMyBP-C peptide with an internal insertion therein, the internal insertion comprising a protease recognition site adjacent to and 5′ to a SpyTag peptide, wherein the internal insertion is within or between C7 and C8 domains of cMyBP-C;
b. isolating myocytes from the host;
c. introducing to the myocytes a protease that cleaves the first cMyBP-C protein at the protease recognition site to remove its N-terminus and expose the SpyTag peptide;
d. introducing a recombinant cMyBP-C N-terminus, the recombinant cMyBP-C N-terminus being at least a portion of cMyBP-C with a SpyCatcher peptide at its C-terminus;
wherein the SpyCatcher peptide of the cMyBP-C N-terminus binds to the SpyTag peptide of the first recombinant cMyBP-C to produce a second recombinant cMyBP-C protein according to claim 3 .
9. A motility assay platform comprising: a coverslip with a thick filament disposed thereon, wherein the thick filament comprises a recombinant cMyBP-C protein according to claim 3 .