Methods for the formation of disulphide bonds
The present invention relates to methods for the formation of inter-molecular disulphide bonds, including (poly)peptides/proteins, nucleic acids, vectors, host cells and bacteriophages used in these methods. Furthermore the invention relates to the use of this method for the improved display of (poly)peptides/proteins on the surface of bacteriophage particles.
1. A method for increasing the display rates of an antibody fragment on the surface of bacteriophage particle, comprising
(a) artificially introducing a first cysteine residue within ten amino acids of the N-terminus of a coat protein of a bacteriophage and artificially introducing a second cysteine residue within fifteen amino acids of the C-terminus of the antibody fragment,
(b) artificially introducing lysine or arginine amino acid within five amino acids of the artificially introduced first and/or second cysteine residue of step (a),
wherein said lysine or arginine amino acid positively affects the reactivity of at least one of said artificially introduced cysteine residues,
(c) causing or allowing the attachment of the coat protein to the antibody fragment,
wherein said attachment is caused by the formation of a disulphide bond between the first cysteine residue comprised in said coat protein and the second cysteine residue comprised in said antibody fragment.
2. The method of claim 1 , wherein said coat protein of a bacteriophage and said antibody fragment are expressed and assembled in an appropriate host cell.
3. The method of claim 1 , wherein said disulphide bond is formed in the periplasmatic space of a host cell.
4. The method of claim 1 , wherein said disulphide bond is a intermolecular disulphide bond.
5. The method of claim 1 , wherein said amino acid of step (b) is lysine.
6. The method of claim 1 , wherein said protein coat comprises a truncated variant of a wild type coat protein of a bacteriophage, wherein said truncated variant comprises at least that part of said wild type coat protein causing the incorporation of said coat protein into the protein coat of the bacteriophage particle.
7. The method of claim wherein said protein coat comprises a variant of a wild type coat protein of a bacteriophage, wherein said variant is capable of being incorporated into the protein coat of the bacteriophage particle.
8. The method of claim 6 , wherein said first cysteine residue has been artificially introduced into said truncated variant of a wild type coat protein of a bacteriophage.
9. The method of claim 7 , wherein said first cysteine residue has been artificially introduced into a variant of a wild type coat protein of a bacteriophage.
10. The method of claim 1 , wherein said bacteriophage particle is a bacteriophage particle of a filamentous bacteriophage.
11. The method of claim 1 , wherein said protein coat of a bacteriophage particle is or is derived from the wild type coat protein pIII.
12. The method of claim 1 , Wherein said protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIX.
13. The method of claim 1 , wherein said antibody fragment is a scFv or Fab fragment.