Process for extracellular secretion of Brazzein

A culture medium for manufacturing a Brazzein peptide, the culture medium comprising:

• a nutrient broth;
• and
• an E. coli cell, the E. coli cell comprising a recombinant nucleic acid, wherein the recombinant nucleic acid comprises:
• (i) a nucleic acid sequence encoding the Brazzein peptide,
• (ii) a nucleic acid sequence encoding a signal leader amino acid sequence,
• wherein the signal leader amino acid sequence is fused at a 5′ end of the Brazzein peptide, and
• wherein the signal leader amino acid sequence is operable to direct extracellular secretion of the Brazzein peptide, and
• (iii) an expression control sequence operable to direct expression of the nucleic acid sequence encoding the signal leader amino acid sequence.

A process of secreting a Brazzein peptide into a nutrient broth, the process comprising:

• culturing an E. coli cell comprising a recombinant nucleic acid in the nutrient broth to generate an E. coli culture, wherein the recombinant nucleic acid comprises:
• (i) a nucleic acid sequence encoding the Brazzein peptide,
• (ii) a nucleic acid sequence encoding a signal leader amino acid sequence,
• wherein the signal leader amino acid sequence is fused at a 5′ end of the Brazzein peptide, and
• wherein the signal leader amino acid sequence is operable to direct extracellular secretion of the Brazzein peptide, and
• (iii) an expression control sequence operable to direct expression of the nucleic acid sequence encoding the signal leader amino acid sequence;
• and
• activating the expression control sequence.

A process for isolating a Brazzein peptide from a nutrient broth, the process comprising:

• culturing an E. coli cell comprising a recombinant nucleic acid in the nutrient broth to generate an E. coli culture, wherein the recombinant nucleic acid comprises:
• (i) a nucleic acid sequence encoding a Brazzein peptide,
• (ii) a nucleic acid sequence encoding a signal leader amino acid sequence fused to a 5′ end of the nucleic acid sequence encoding the Brazzein peptide,
• wherein cellular translation generates the Brazzein peptide having the signal leader amino acid sequence located at a 5′ end,
• wherein the signal leader amino acid sequence is operable to direct extracellular secretion of the Brazzein peptide, and
• (iii) an expression control sequence operable to direct expression of the nucleic acid sequence encoding the signal leader amino acid sequence;
• activating the expression control sequence;
• and
• separating the E. coli culture to form a cell pellet and a supernatant, wherein the supernatant comprises the Brazzein peptide.