Cysteine cross-linked structural peptides

A method comprising:

• a) providing a host cell comprising an expressible genetic construct encoding an insoluble fusion peptide comprising at least one first portion and at least one second portion, wherein said first portion comprises a multi-functional solubility tag (MST) and said second portion comprises a peptide of interest (POI);
• wherein the multi-functional solubility tag (MST) has the general formula of:
• SEQ ID NO:
• 1—Spacer−[[SEQ ID NO:
• 1]−[Spacer]m]n or (i)
• SEQ ID NO:
• 2—Spacer−[[SEQ ID NO:
• 2]−[Spacer]m]n;
• (ii)
• wherein
• SEQ ID NO:
• 1 is Xaa1-Gln-[Xaa2]p-[Phe-Xaa3-Xaa4-Xaa5]s-Phe-Xaa6-[Xaa7]q-[Gln]r;
• and
• SEQ ID NO:
• 2 is Xaa1-Gln-Xaa8-[Xaa4-Xaa8]s-Phe-[Glu-Gln-Gln]r;
• wherein
• Xaa1=Gln or His;
• Xaa2=Gln, Arg, His, or Lys;
• Xaa3=Gln, His, Lys, Arg, or Glu;
• Xaa4=Trp or Phe;
• Xaa5=Gln, His, Lys, Arg or Glu;
• Xaa6=Glu, Gln, or Arg;
• Xaa7=Gln or Lys;
• Xaa8=Asp, Glu, Gln, His, Lys, or Arg;
• p, q, and r are independently 0 or 1;
• s is an integer ranging from 1 to 5;
• n is an integer ranging from 2 to 10;
• m=n or n−1;
• and
• Spacer=a peptide linker ranging from 1 to 100 amino acids in length;
• wherein said spacer comprises at least one cross-linkable cysteine residue;
• b) growing the microbial host cell under conditions whereby the insoluble fusion peptide is produced within the host cell in the form of at least one inclusion body;
• c) recovering the insoluble fusion peptide from the microbial host cell;
• d) subjecting the recovered insoluble fusion peptide of (c) to aqueous reducing conditions have a pH of at least 10 for a period of time sufficient to solubilize the insoluble fusion peptide and reduce cross-linkable cysteine residues;
• whereby an aqueous solution comprising a population of reduced fusion peptides is produced;
• e) contacting the aqueous solution comprising the population of reduced fusion peptides of (d) with a first target material whereby a mixture is formed;
• f) altering the pH of the mixture of (e) whereby at least a portion of the population of reduced fusion peptides non-covalently associates with the first target material forming fusion peptide-first target material complexes;
• wherein the association between the fusion peptides and the first target material is dependent upon, or enhanced by, the presence of the multi-functional solubility tag;
• g) subjecting the fusion peptide-first target material complexes of (f) to oxidizing conditions whereby the cysteine residues cross-link;
• and
• h) optionally recovering the product of step (g).

An affinity media having a solid support comprising an effective amount of a fusion peptide coating, said fusion peptide having a first portion and a second portion, wherein said first portion comprises a multi-functional solubility tag (MST) that is bound to the solid support and said second portion of the fusion peptide has an affinity for a target material;

• wherein the multi-functional solubility tag has the general formula:
• SEQ ID NO:
• 1—Spacer−[[SEQ ID NO:
• 1]−[Spacer]m]n or a)
• SEQ ID NO:
• 2—Spacer−[[SEQ ID NO:
• 2]−[Spacer]m]n;
• b)
• wherein
• SEQ ID NO:
• 1 is Xaa1-Gln-[Xaa2]p-[Phe-Xaa3-Xaa4-Xaa5]s-Phe-Xaa6-[Xaa7]q-[Gln]r;
• and
• SEQ ID NO:
• 2 is Xaa1-Gln-Xaa8-[Xaa4-Xaa8]s-Phe-[Glu-Gln-Gln]r;
• wherein
• Xaa1=Gln or His;
• Xaa2=Gln, Arg, His, or Lys;
• Xaa3=Gln, His, Lys, Arg, or Glu;
• Xaa4=Trp or Phe;
• Xaa5=Gln, His, Lys, Arg or Glu;
• Xaa6=Glu, Gln, or Arg;
• Xaa7=Gln or Lys;
• Xaa8=Asp, Glu, Gln, His, Lys, or Arg;
• p, q, and r are independently 0 or 1;
• s is an integer ranging from 1 to 5;
• n is an integer ranging from 1 to 10;
• m=n or n−1;
• and
• Spacer=a peptide linker ranging from 1 to 100 amino acids in length, wherein said spacer comprises at least one cross-linkable cysteine residue.