Methods for the expression of peptides and proteins

A method for production of a recombinant peptide (PeOI) or protein of interest (PrOI), wherein the method comprises:

• (a) introducing a nucleic acid molecule encoding a fusion protein comprising at least one PeOI or PrOI, and at least one allocrite of a type 1 secretion system (TISS) or a fragment thereof, into a host cell, wherein the host cell does not express one or more of a heterologous ATP-binding cassette (ABC) transporter of the TISS, a heterologous membrane fusion protein (MFP) of the TISS, and a heterologous outer membrane protein (OMP) of the TISS, and wherein the allocrite of the T1 SS or fragment thereof (i) comprises at least one GG repeat of a consensus sequence that comprises the amino acid sequence set forth in SEQ ID NO:67, (ii) causes expression of the fusion protein in an insoluble, denatured form of inclusion bodies (IB), and (iii) promotes refolding of the insoluble, denatured form of the fusion protein when the fusion protein is contacted with a refolding buffer that comprises at least 0.01 mM of an earth alkaline metal ion;
• (b) cultivating the host cell under conditions that allow expression of the fusion protein, wherein the fusion protein is expressed in the insoluble, denatured form of inclusion bodies (IB);
• (c) isolating the inclusion bodies (IB) of step (b) from said host cells;
• (d) isolating the insoluble, denatured form of the fusion protein from said IB of step (c);
• and
• (e) subjecting the insoluble, denatured form of the fusion protein isolated in step (d) to conditions that allow the PeOI or PrOI to refold into a soluble, functional three-dimensional conformation, by contacting the fusion protein with said refolding buffer that comprises at least 0.01 mM of an earth alkaline metal ion.