Cysteine cross-linked structural peptides

1. 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 -[Gin] 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 3 to 60 amino acids in length; wherein said spacer comprises at least one cross-linkable cysteine residue;

b) growing the microbial host cell 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 having a pH of at least 10 for a period of time 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 the population of reduced fusion peptides non-covalently binds with the first target material forming fusion peptide-first target material complexes; wherein the binding 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).

2. The method of claim 1 wherein the multi-functional solubility tags are less than 400 amino acids in length.

3. The method of claim 1 wherein the reducing conditions comprises a pH of at least 11.

4. The method of claim 1 wherein the reducing conditions comprises at least one reducing agent.

5. The method of claim 1 wherein the step of altering of the pH of the aqueous mixture is reducing the pH.

6. The method of claim 5 comprising reducing the pH to 8 or less.

7. The method of claim 5 comprising reducing the pH by dialysis.

8. The method of claim 1 comprising subjecting the fusion peptide-first target material complex to oxidizing conditions in step (g) by dialysis.

9. The method of claim 1 wherein the first target material is a particle having an average particle size ranging from 3 nm to 10 μm as measured by a light scattering method.

10. The method of claim 1 wherein the first portion of the fusion peptide forms a hydrogel and the second portion has affinity for a human tissue or cell surface receptor.

11. The method of claim 10 wherein the second portion having affinity for a human tissue or cell surface receptor comprises at least one arginine-glycine-aspartic acid (RGD) peptide.

12. The method of claim 1 wherein the number of cross-linkable cysteine residues in said MST is at least 3.