Optimization of the production of gurmarin, a sweet-taste-suppressing protein, secreted by the methylotrophic yeast Pichia pastoris.
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.
Centre des Sciences du Goût et de l'Alimentation, UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne, 21000, Dijon, France.
Maud
M
Sigoillot
Anne
A
Brockhoff
Ewen
E
Lescop
Nicolas
N
Poirier
Wolfgang
W
Meyerhof
Loïc
L
Briand
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D021382:Protein Sorting Signals; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010940:Plant Proteins; D021382:Protein Sorting Signals; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C431926:taste receptors, type 1; C075206:Gurmarin protein, Gymnema sylvestre