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CN-122003432-A - Recombinant polypeptides comprising a metal binding motif

CN122003432ACN 122003432 ACN122003432 ACN 122003432ACN-122003432-A

Abstract

The present disclosure relates generally to a recombinant polypeptide comprising an IgG binding domain derived from streptococcal protein G (SpG) and a metal binding motif, wherein the recombinant polypeptide has a first, target-binding conformation in the presence of a metal ion and a second, non-target-binding conformation in the absence of a metal ion, wherein the recombinant polypeptide is configured to bind to a Fab region of IgG in the first, target-binding conformation. The disclosure also relates to ligands comprising the recombinant polypeptides, chromatography matrices comprising ligands coupled to matrices, and methods of purifying sample proteins using chromatography matrices. Furthermore, the disclosure relates to nucleic acids encoding the recombinant polypeptides, and expression systems comprising the nucleic acids.

Inventors

  • SOPHIA HOBER
  • Marlin jonson

Assignees

  • 霍伯生物技术公司

Dates

Publication Date
20260508
Application Date
20241021
Priority Date
20231025

Claims (16)

  1. 1. A recombinant polypeptide comprising an IgG binding domain derived from streptococcal protein G (SpG) and a metal binding motif, wherein the recombinant polypeptide has a first, target-binding conformation in the presence of a metal ion and a second, non-target-binding conformation in the absence of a metal ion, wherein the recombinant polypeptide is configured to bind IgG in the first, target-binding conformation.
  2. 2. The recombinant polypeptide of claim 1, wherein the IgG binding domain is domain C1 or domain C2.
  3. 3. Recombinant polypeptide according to claim 1 or claim 2, wherein the metal binding motif is inserted into the N-terminus of the alpha helix of the IgG binding domain, preferably between the second beta strand and the alpha helix of the IgG binding domain.
  4. 4. The recombinant polypeptide according to any one of the preceding claims, wherein the recombinant polypeptide is configured to bind to a Fab region of the IgG in the first, target-binding conformation.
  5. 5. The recombinant polypeptide according to any one of the preceding claims, wherein the metal binding motif is a calcium binding motif, and wherein the metal ion is a calcium ion.
  6. 6. The recombinant polypeptide according to any one of the preceding claims, wherein the metal binding motif is a metal binding loop comprising 10 to 14 amino acids, preferably 12-13 amino acids.
  7. 7. The recombinant polypeptide according to any one of the preceding claims, wherein the metal binding motif is defined by the sequence: X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 (SEQ ID NO:1), Wherein the method comprises the steps of X 4 is aspartic acid (D), X 5 is alanine (A), arginine (R), glutamic acid (E), lysine (K), serine (S), threonine (T), leucine (L) or valine (V), X 6 is aspartic acid (D) or asparagine (N), X 7 is arginine (R), asparagine (N), glycine (G) or lysine (K), X 8 is asparagine (N), aspartic acid (D) or serine (S), preferably aspartic acid (D), X 9 is glycine (G), X 10 is arginine (R), glycine (G), lysine (K), phenylalanine (F), threonine (T), histidine (H) or tyrosine (Y), preferably phenylalanine (F), tyrosine (Y) or arginine (R), X 11 is isoleucine (I), leucine (L) or valine (V), X 12 is asparagine (N), aspartic acid (D), glutamic acid (E), serine (S) or threonine (T), preferably asparagine (N) or aspartic acid (D), X 13 is alanine (A), phenylalanine (F), threonine (T) or tyrosine (Y), X 14 is aspartic acid (D), glutamic acid (E) or lysine (K), X 15 is aspartic acid (D) or glutamic acid (E), preferably aspartic acid (D), X 16 is alanine (A), leucine (L), phenylalanine (F) or valine (V).
  8. 8. The recombinant polypeptide according to any one of the preceding claims, further comprising a linker upstream of the metal binding motif, wherein the linker comprises 1 to 4 amino acids, preferably 2 to 3 amino acids.
  9. 9. The recombinant polypeptide according to claim 8 when dependent on claim 7, wherein said metal binding motif comprising said linker is defined by: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 (SEQ ID NO: 2), Wherein the method comprises the steps of X 1 is serine (S), threonine (T), glycine (G), proline (P) or is absent, X 2 and X 3 are selected from serine (S), threonine (T), glycine (G) or proline.
  10. 10. A recombinant polypeptide according to any one of the preceding claims, wherein the metal binding motif is defined by a sequence selected from any one of SEQ ID NOs 6-19 or a corresponding sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence homology with any one of SEQ ID NOs 6-19.
  11. 11. The recombinant polypeptide according to any one of the preceding claims, wherein the IgG binding domain comprises at least one mutation, wherein the at least one mutation is a substitution of glutamic acid (E) with glycine (G) at the first amino acid residue of the alpha helix of the IgG binding domain.
  12. 12. A ligand comprising the recombinant polypeptide of any one of claims 1-11.
  13. 13. A chromatography matrix comprising the ligand of claim 12 coupled to the matrix.
  14. 14. A method for purifying a sample protein, comprising: a) The chromatography matrix according to claim 13 is provided, B) Adding a metal ion to the chromatography matrix, C) Adding a sample comprising the target protein, D) Elution of the bound target protein is preferably performed at a pH of 5 to 8.
  15. 15. A nucleic acid encoding the recombinant polypeptide of any one of claims 1-11.
  16. 16. An expression system comprising the nucleic acid of claim 15.

Description

Recombinant polypeptides comprising a metal binding motif Technical Field The present disclosure relates generally to a recombinant polypeptide comprising an IgG binding domain derived from Streptococcus (Streptococcus) protein G (SpG) and a metal binding motif. The disclosure also relates to ligands comprising recombinant polypeptides, chromatography matrices comprising ligands coupled to matrices, and methods of purifying sample proteins using chromatography matrices. The disclosure also relates to nucleic acids encoding the recombinant polypeptides and expression systems comprising the nucleic acids. Background Antibodies, also known as immunoglobulins (Ig), are commonly used in the field of bioscience and are an important class of biotherapeutic drugs. Antibodies are useful in the treatment of a variety of diseases, including allergies, autoimmune diseases, and cancers. An antibody is a relatively large Y-shaped protein and comprises two antigen binding fragments (Fab) and one crystallizable fragment (Fc) forming a Y-shaped backbone. Immunoglobulin G (IgG) is the predominant antibody type found in blood and extracellular fluids and represents the majority of antibody-based immunity against invading pathogens. Bacteria such as staphylococci and streptococci have evolved to express surface proteins that have an inherent affinity for immunoglobulins (Ig) with a broad range of species specificities as a means of evading immune responses. Many engineering works have demonstrated that these proteins can be used as model systems because they are well characterized in terms of structural properties, interactions and thermodynamic stability. In addition, such Ig-binding proteins are commonly used in affinity chromatography applications to recover immunoglobulins (Ig) from different samples. Staphylococcus aureus (Staphylococcus aureus) protein a (SpA) is a widely used affinity chromatography ligand. SpA binds with high affinity to the Fc region of immunoglobulins. Various attempts have been made to date by recombinant techniques to increase the binding capacity of protein a and its domains. In addition, various methods have been developed to reduce the sensitivity of protein A to alkaline pH, which is commonly used in elution steps of affinity chromatography. EP3523318B1 relates to an IgG binding polypeptide mutant derived from the Z domain of SpA domain B, which mutant binds to the Fc region of an immunoglobulin and comprises a metal binding motif inserted between helix 2 and helix 3 of the Z domain. The IgG-binding polypeptide mutants allow elution of IgG by removal of calcium at ph5.5 to 7.0. Streptococcal Protein G (SPG) is another protein with affinity for immunoglobulins. SPG typically binds to Fc fragments of IgG from a variety of mammalian species. SPG consists of a repeating arrangement of domains, with the C-terminal domains (C1, C2 and C3) responsible for IgG binding and the N-terminal domain binding to human serum albumin. The tertiary structure and amino acid composition of Streptococcal Protein G (SPG) are significantly different from those of protein a. Thus, these proteins have different binding characteristics. For example, SPG may be used to purify IgG that binds poorly to protein A. With the rapid growth of the therapeutic antibody market, there is an increasing demand for new and improved ligands. Thus, igG-binding polypeptides derived from Streptococcal Protein G (SPG) will become important for the recovery and isolation of immunoglobulins or fragments thereof. Accordingly, there is a need to provide an improved SPG that is capable of selectively binding immunoglobulins (IgG) with high affinity and which can be used to recover and isolate IgG from target samples. Disclosure of Invention In view of the above, it is an object of the present disclosure to provide improvements to IgG-binding polypeptides derived from streptococcal protein G. According to a first aspect of the present disclosure there is provided a recombinant polypeptide comprising an IgG binding domain derived from Streptococcal Protein G (SPG) and a metal binding motif, wherein the recombinant polypeptide has a first, target-binding conformation in the presence of a metal ion and a second, non-target-binding conformation in the absence of a metal ion, wherein the recombinant polypeptide is configured to bind IgG in the target-binding conformation. The inventors have found that metal binding motifs can be successfully incorporated into IgG binding domains derived from SPG and thereby provide a recombinant metal-dependent protein G binding domain. Upon binding to the metal ion, the recombinant polypeptide can transition from an inactive state (i.e., a non-target binding conformation) to a functional state (i.e., a target binding conformation). The inventors have isolated a variety of polypeptides whose inherent target affinity for antibodies and antibody fragments has been converted to metal ion dependence by engineering efforts