Methods and compositions for production of recombinant peptides

A method for solubilizing and recovering, in bioactive and isolated form, a target peptide from a host organism in which the target peptide is present in insoluble form, which comprises:

• (a) disrupting the host cell to produce a lysate;
• (b) precipitating said lysate
• (c) recovering lysate precipitate containing the polypeptide
• (d) resuspending the lysate precipitate in a denaturant-free non-buffered solubilization solution to produce a solubilization preparation that comprises
• 1) hydrogen chloride at between about 10 and about 20 mM and
• 2) polypeptide at between about 1 and about 4 mg precipitate per ml solubilization solution, and
• 3) pH of between about 2.0 and about 3.0;
• and
• (d) recovering bioactive the target peptide as supernatant from the solubilization preparation of (c).

A method for isolating recombinant proteins comprising:

• providing a non-buffered solution that comprises a stabilizing compound and hydrogen chloride between about 10 and about 20 mM;
• producing a protein solution by adding to the non-buffered solution a recombinant polypeptide between about 1 and about 4 mg polypeptide per ml non-buffered solution, wherein the protein solution has a pH of between about 2.0 and about 3.0;
• increasing the pH of the protein solution to between about 4 and 5 using 1N NaOH;
• centrifuging the protein solution and recovering precipitate-free supernatant;
• and adjusting the pH of the supernatant to between about pH 9 and 10.5 with 1N NaOH
• retaining the supernatant comprising isolated target protein at least about 10% more pure than the isolated target protein in aggregate form.

A method for isolating recombinant proteins comprising:

• providing a non-buffered solution that comprises a stabilizing compound and sodium hydroxide between about 8 and about 10 mM;
• producing a protein solution by adding to the non-buffered solution a recombinant polypeptide between about 1 and about 4 mg polypeptide per ml non-buffered solution, wherein the protein solution has a pH of between about 9 and about 11.2;
• lowering the pH of the protein solution to between about 4 and 5 using 1N NaOH;
• centrifuging the protein solution and recovering precipitate-free supernatant;
• and
• adjusting the pH of the supernatant to between about pH 9 and 10.5 with 1N NaOH
• retaining the supernatant comprising isolated target protein at least about 10% more pure than the isolated target protein in aggregate form.

A method for preparing bioactive recombinant polypeptide in a chaotrope-containing solution, comprising:

• decreasing the concentration of the chaotropic agent in the chaotrope-containing solution by dialyzing the chaotrope-containing solution against a renaturing buffer of pH between about 9 and about 11.2 and buffer concentration between about 10 and about 50 mM, wherein the renaturing buffer further comprises a stabilizing compound;
• chromatographically purifying the protein;
• and
• dialyzing the isolated protein against an aqueous stabilization buffer comprising a stabilizing compound.