Method for producing a condensed adhesive phase of silk fusion proteins

A method for producing a condensed phase of a silk fusion protein, the method comprising the steps of:

• a) preparing a solution of a silk fusion protein in an aqueous medium, wherein said silk fusion protein is isolated from a recombinant production host and comprises a silk-like protein sequence and two separate non-silk terminal module sequences flanking said silk-like protein sequence;
• b) concentrating said silk fusion protein in said aqueous medium until a liquid phase separation occurs at about 1% w/v;
• c) collecting a protein containing phase of said aqueous medium obtained in step (b);
• d) repeating steps (b) and (c) with the protein containing phase obtained in step (c) until a protein concentration of about 20-45% w/v is reached;
• e) optionally separating aggregates of the silk fusion protein from soluble fusion proteins obtained in step (d) and removing the aggregates from the protein containing phase;
• and
• f) concentrating the protein containing phase obtained in step (d) or (e) to form a concentrate with a final protein concentration of about 60-80% w/v,
• wherein the steps (a) to (f) are performed so that said silk fusion protein is not precipitated and subsequently dissolved in said aqueous medium,
• wherein said non-silk terminal module sequences flanking said silk-like protein sequence comprise cellulose binding modules (CBMs), SpyCatcher domains, gamma-crystallin D domains, tenth type III modules of fibronectin or a mixed pair thereof, and
• wherein said cellulose binding modules or said SpyCatcher domains flanking said silk-like protein sequence are linked to said silk-like protein sequence by a linker sequence, and
• wherein said linker sequence is selected from the group consisting of:
• a C-terminal linker of SEQ ID NO:6 and a N-terminal linker of SEQ ID NO:5.