Affinity purification, proximity-based sortase ligation, and detection of proteins with precursor peptides and b1 proteins from lasso peptide biosynthesis systems
AssigneesThe Trustees of the University of Pennsylvania, AlphaThera LLC
InventorsAndrew TSOURKAS, Yi Shu
Life of this patent
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Abstract
The invention relates to the use of precursor peptides and B1 proteins from lasso peptide biosynthesis systems for affinity purification, proximity-based sortase-mediated protein purification and ligation, and detection of fusion proteins. For proximity-based sortase-mediated protein purification and ligation, the invention relates to techniques that link protein purification with conjugation to other agents, including therapeutic agents, imaging agents, or linkers.
Claims72
A conjugate protein composition comprising a first fusion protein and a second fusion protein, wherein:
- (i) the first fusion protein comprises a protein of interest in series with a sortase recognition motif and a first binding pair member;
- wherein:
- said protein of interest is N-terminal and connected via a linker to said sortase recognition motif and said sortase recognition motif is N-terminal and connected via a linker to the first binding pair member;
- or
- said first binding pair member is N-terminal and connected via a linker to said sortase recognition motif and said sortase recognition motif is N-terminal and connected via a linker to said protein of interest;
- and
- (ii) the second fusion protein comprises a second binding pair member in series with a sortase and optionally a first affinity tag having a selective affinity for a first affinity tag resin, wherein:
- said second binding pair member is N-terminal and connected via a linker to said sortase;
- or
- said sortase is N-terminal and connected via a linker to said second binding pair member;
- and when said first affinity tag is present, said first affinity tag is connected via a linker either N-terminal or C-terminal to said second binding pair member in series with said sortase;
- wherein the binding pair members comprise a leader peptide and a B1 protein pair from a lasso peptide biosynthesis system that can form a heterodimer.
The conjugate protein composition of any one of the preceding claims, wherein the first affinity tag is selected from the group consisting of a histidine tag (His tag), a chitin-binding domain, a calmodulin tag, a polyglutamate tag, a maltose binding protein, glutathione-S-transferase, an S-tag, SBP-tag, Strep-tag, Strep-tag II, green fluorescent protein-tag, thioredoxin tag, Nus-tag, Fc-tag, Halo-tag, FLAG-tag, V5-tag, VSV-tag, Xpress tag, E-tag, Myc-tag, HA-tag, Softag, and NE-tag, biotin (via biotin ligase), BirA, AviTag, BCCP, SpyTag, SpyCathcher, SnoopTag, and SnoopCatcher.
The conjugate protein composition of any one of the preceding claims, wherein said first fusion protein further comprises a second affinity tag having a selective affinity for a second affinity tag resin.
The conjugate protein composition of any one of the preceding claims, wherein said sortase is selected from the group consisting of sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), and variants thereof.
The conjugate protein composition of any one of the preceding claims, wherein the sortase recognition motif is selected from the group consisting of LPXTG (SEQ ID NO:
- 1), LPKTG (SEQ ID NO:
- 2), LPATG (SEQ ID NO:
- 3), LPNTG (SEQ ID NO:
- 4), LPETG (SEQ ID NO:
- 5), LPXAG (SEQ ID NO:
- 6), LPNAG (SEQ ID NO:
- 7), LPXTA (SEQ ID NO:
- 8), LPNTA (SEQ ID NO:
- 9), LGXTG (SEQ ID NO:
- 10), LGATG (SEQ ID NO:
- 11), IPXTG (SEQ ID NO:
- 12), IPNTG (SEQ ID NO:
- 13), IPETG (SEQ ID NO:
- 14), and NPQTN (SEQ ID NO:
- 15), LAXTG (SEQ ID NO:
- 16), LPXSG (SEQ ID NO:
- 17), LSETG (SEQ ID NO:
- 18), LPXCG (SEQ ID NO:
- 19), LPXAG (SEQ ID NO:
- 20), and XPETG (SEQ ID NO:
- 21).
The conjugate protein composition of any one of the preceding claims, wherein the linkers are flexible GS-rich linkers.
The conjugate protein composition of any one of the preceding claims, further comprising said first affinity tag resin to which said first affinity tag selectively binds.
The conjugate protein composition of any one of the preceding claims, wherein said protein of interest is an antibody or antigen-binding fragment thereof.
A conjugate protein composition comprising a first fusion protein and a second fusion protein, wherein:
- (i) the first fusion protein comprises a protein of interest in series with and connected via a linker to a first binding pair member;
- and
- (ii) the second fusion protein comprises a second binding pair member optionally in series with and connected via a linker to a first affinity tag having a selective affinity for a first affinity tag resin;
- wherein the binding pair members comprise a leader peptide and a B1 protein pair from a lasso peptide biosynthesis system that can form a heterodimer.
A vector encoding said first fusion protein and/or the second fusion protein of any one of claims 36-54.
A method of detecting a protein of interest, said method comprising;
- (a) providing a protein of interest connected via a linker to a first binding pair member;
- (b) contacting the first binding pair member with a second binding pair member wherein the second binding pair member is attached to an imaging agent or molecular reporter;
- and
- (c) imaging or detecting said imaging agent or molecular reporter. wherein the first and second binding pair members comprise a leader peptide and a B1 protein pair from a lasso peptide biosynthesis system that can form a heterodimer.
Dependencies are inferred from the claim text.