Method of preparing cysteine mutants of human recombinant GM-CSF

A method for preparing a recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) protein, wherein the GM-CSF protein is a human GM-CSF cysteine mutein comprising a mutein selected from the group consisting of a cysteine residue added preceding the first amino acid of the mature protein, a cysteine residue added following the last amino acid of the protein and a cysteine residue substituted for an amino acid selected from the group consisting of A1, A3, S5, S7, N27, T32, A33, E51, R67, S69, E93, T94, T98, Q99, T102, E123, V125, Q126, and E127;

• wherein the method comprises the steps of:
• (a) obtaining an Escherichia coli (E. coli) host cell transformed with a plasmid encoding a GM-CSF protein fused to the E. coli STII signal sequence;
• (b) growing the E. coli host cell under conditions that cause the host cell to express the GM-CSF protein and secrete the GM-CSF protein into the E. coli periplasm;
• (c) lysing the cells;
• (d) isolating the insoluble GM-CSF protein from the lysed cells;
• (e) denaturing and reducing the insoluble GM-CSF protein;
• and
• (f) refolding the GM-CSF protein into a biologically active form.