Centre des Sciences du Goût et de l'Alimentation, UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne, 21000, Dijon, France.
Appl Microbiol Biotechnol. 2012 Dec;96(5):1253-63. doi: 10.1007/s00253-012-3897-3. Epub 2012 Feb 4.
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.
Gurmarin 是一种 35 个氨基酸组成的多肽，已知它能选择性地抑制啮齿动物对甜味物质的反应，而不影响对其他基本味觉刺激物（如 NaCl、HCl 和奎宁）的反应。在这里，我们报告了使用甲醇营养酵母毕赤酵母异源表达 gurmarin。gurmarin 使用 α 因子前导序列分泌到缓冲的最小培养基中，而不使用酿酒酵母的 EAEA 间隔肽，并受甲醇诱导的醇氧化酶启动子的控制。我们发现，gurmarin 在酵母培养物中积累，在 4 天的表达期内达到每升培养物 5 毫克。为了比较生产水平和信号肽加工，gurmarin 的 N 端氨基酸被谷氨酸残基取代。该构建体导致 gurmarin 分泌水平提高了 6 倍，每升培养物产生 30 毫克纯化蛋白。两种构建体产生的纯化重组 gurmarin 均使用质谱进行了表征。圆二色性和 NMR 光谱表明，重组 gurmarin 正确折叠并具有二级和三级结构。我们还通过在人胚胎肾 HEK293T 细胞中的功能性表达证实了它抑制大鼠异源二聚体甜味 T1R2/T1R3 受体的能力。毕赤酵母中获得的高浓度完全活性 gurmarin 使该表达系统在发酵罐生长和味觉受体和 gurmarin 功能的药理学研究方面具有吸引力。
