Optimization of the production of gurmarin, a sweet-taste-suppressing protein, secreted by the methylotrophic yeast Pichia pastoris

• Published in: Applied microbiology and biotechnology 2012-12, Vol.96 (5), p.1253-1263
• Main Authors: Sigoillot, Maud, Brockhoff, Anne, Lescop, Ewen, Poirier, Nicolas, Meyerhof, Wolfgang, Briand, Loïc
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analysis; Animals; Base Sequence; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical Sciences; Chemical synthesis; Circular Dichroism; Cloning; Gene Expression; Genetic recombination; Life Sciences; Magnetic Resonance Spectroscopy; Mass Spectrometry; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Nuclear magnetic resonance spectroscopy; Organic chemistry; Oxidases; Peptides; Pichia - genetics; Pichia pastoris; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - secretion; Plasmids; Polypeptides; Promoter Regions, Genetic; Protein Conformation; Protein Folding; Protein Sorting Signals; Proteins; Rats; Receptors, G-Protein-Coupled - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - secretion; Saccharomyces cerevisiae; Sodium chloride; Studies; Taste; Yeast; Yeasts
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-3897-3

Gurmarin, a 35-residue polypeptide, is known to selectively inhibit responses to sweet substances in rodents without affecting responses to other basic taste stimuli, such as NaCl, HCl, and quinine. Here, we report the heterologous expression of gurmarin using the methylotrophic yeast Pichia pastoris . Gurmarin was secreted into the buffered minimal medium using the α-factor preprosequence without the EAEA spacer peptide of Saccharomyces cerevisiae and was under the control of the methanol-inducible alcohol oxidase promoter. We found that gurmarin accumulated in the yeast culture medium reaching 5 mg per liter of culture over an expression period of 4 days. To compare the production level and the signal peptide processing, the N-terminal amino acid of gurmarin was substituted by a glutamic acid residue. This construct resulted in a 6-fold increase in the level of gurmarin secretion leading to 30 mg of purified protein per liter of culture. Purified recombinant gurmarin resulting from both constructs was characterized using mass spectrometry. Circular dichroism and NMR spectroscopy revealed that recombinant gurmarin was properly folded and had secondary and tertiary structures. We also confirmed its capability to inhibit the rat heterodimeric sweet taste T1R2/T1R3 receptor by functional expression in human embryonic kidney HEK293T cells. The high level of fully active gurmarin obtained in P. pastoris makes this expression system attractive for fermentor growth and pharmacological investigations of taste receptor and gurmarin functions.