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

Autor: Nicolas Poirier, Wolfgang Meyerhof, Loïc Briand, Ewen Lescop, Anne Brockhoff, Maud Sigoillot
Přispěvatelé: Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)
Jazyk: angličtina
Rok vydání: 2012
Předmět:
Protein Folding
Magnetic Resonance Spectroscopy
Protein Conformation
Molecular Sequence Data
Gene Expression
Protein Sorting Signals
Biology
Signal peptide processing
Applied Microbiology and Biotechnology
Mass Spectrometry
Pichia
Receptors
G-Protein-Coupled
Pichia pastoris
03 medical and health sciences
0302 clinical medicine
Taste receptor
Animals
Amino Acid Sequence
Promoter Regions
Genetic
Plant Proteins
030304 developmental biology
Gurmarin
chemistry.chemical_classification
0303 health sciences
Base Sequence
[CHIM.ORGA]Chemical Sciences/Organic chemistry
Circular Dichroism
General Medicine
biology.organism_classification
Recombinant Proteins
Yeast
Rats
3. Good health
Amino acid
Biochemistry
chemistry
Heterologous expression
Gymnema sylvestre
030217 neurology & neurosurgery
Biotechnology
Zdroj: Applied Microbiology and Biotechnology
Applied Microbiology and Biotechnology, Springer Verlag, 2012, 96 (5), pp.1253-63. ⟨10.1007/s00253-012-3897-3⟩
ISSN: 0175-7598
1432-0614
Popis: International audience; 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.
Databáze: OpenAIRE
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