Target recognition motifs and uses thereof
View Patent ↗Novel programmable targeting sequences and applications thereof. The targeting sequences can be engineered for binding to proteins, polypeptides, and other macromolecules.
1. An engineered protein or polypeptide, comprising two or more engineered, heterologous Flavobacterium psychrophilum leucine-rich repeats (LRRs) and a trafficking domain, wherein
(a) at least one of the one or more LRRs comprises X 1 (P/G)X 2 (T/S)X 3 FX 4 , wherein:
X 1 is 4 to 6 amino acids in length and comprises non-charged polar amino acids and aliphatic hydrophobic amino acids,
X 2 is 2 to 4 amino acids in length and comprises a charged or non-charged polar amino acid and/or an aliphatic hydrophobic amino acid,
X 3 is 1 to 6 amino acids in length and comprises any amino acid,
X 4 is 1 to 5 amino acids and comprises at least one polar amino acid, and
(b) at least one of the LRRs comprises LX 1 I(P/G)X 2 (T/S)IFC, wherein:
X 1 comprises one or more of T, S, and V, and
X 2 comprises one or more of N, S, and V; and
the trafficking domain comprises a secretion signal peptide and either a secretion system substrate or a member of a specific binding pair.
2. The engineered protein or polypeptide of claim 1 , wherein:
the aliphatic hydrophobic amino acids of X 1 is independently selected from the group consisting of leucine, isoleucine, and valine,
one or more amino acids of X 3 are independently selected from the group consisting of serine, threonine, alanine, and cysteine, or
the polar amino acid of X 2 is independently selected from the group consisting of
asparagine, a serine,
and an aspartate.
3. The engineered protein or polypeptide of claim 1 , wherein one or more amino acids of X 3 are independently selected from the group consisting of charged and non-charged polar amino acids.
4. The engineered protein or polypeptide of claim 1 , wherein one or more amino acids of X 3 are independently selected from the group consisting of aliphatic and aromatic hydrophobic amino acids.
5. The engineered protein or polypeptide of claim 4 , wherein the aliphatic hydrophobic amino acids of X 3 are independently selected from the group consisting of alanine, valine, glycine, and isoleucine.
6. The engineered protein or polypeptide of claim 3 , wherein
the non-charged polar amino acids of X 3 are independently selected from the group consisting of tyrosine, serine, asparagine, and threonine, or
the charged polar amino acids of X 3 are independently selected from the group consisting of aspartate and glutamate.
7. The engineered protein or polypeptide of claim 1 , wherein the at least one polar amino acid of X 4 is independently selected from the group consisting of a charged and a non-charged polar amino acid.
8. The engineered protein or polypeptide of claim 7 , wherein
the charged polar amino acid of X 4 is selected from the group consisting of lysine and arginine, and
the non-charged polar amino acid of X 4 is selected from the group consisting of serine, tyrosine, asparagine, and cysteine.
9. The engineered protein or polypeptide of claim 1 , wherein at least one amino acid of X 1 is independently selected from the group consisting of leucine, serine, threonine, valine, and isoleucine;
wherein at least one amino acid of X 2 is independently selected from the group consisting of asparagine, serine, and valine;
wherein at least one amino acid of X 3 is independently selected from the group consisting of isoleucine, glycine, serine, threonine, and alanine; and
wherein at least one amino acid of X 4 is independently selected from the group consisting of cysteine, serine, isoleucine, and glycine.
10. The engineered protein or polypeptide of claim 1 , wherein the two or more LRRs are independently selected from the group consisting of SEQ ID NOs: 49-69 and 71-87.
11. The engineered protein or polypeptide of claim 1 , wherein
the trafficking domain comprises a Type IX secretion system (T9SS) substrate,
further comprises an amino acid sequence capable of directing clearance from a cell or organism,
the trafficking domain is capable of binding to an Fc receptor, binding to a dendritic cell, or directing antigen processing,
the protein or polypeptide binds to a pathogen,
the protein or polypeptide binds to a virus,
the protein or polypeptide binds to a toxin, or
the protein or polypeptide blocks an activity of a pathogen, virus or toxin.
12. An isolated polynucleotide encoding the engineered protein or polypeptide of claim 1 .
13. A The polynucleotide of claim 12 , wherein the polynucleotide is optimized for expression in an isolated host cell.
14. The polynucleotide of claim 13 , wherein the isolated host cell is a prokaryotic cell.
15. The polynucleotide of claim 13 , wherein the isolated host cell is a eukaryotic cell.
16. The polynucleotide of claim 15 , wherein the eukaryotic cell is a human cell, a mammalian cell, a plant cell, or a yeast cell.
17. The polynucleotide of claim 12 , wherein the polynucleotide comprises a regulatory element.
18. The polynucleotide of claim 17 , wherein the regulatory element is inducible.
19. A vector comprising the polynucleotide of claim 12 .
20. The vector of claim 19 , wherein the vector is a viral vector, a bacteriophage vector, or a plasmid vector.