Genetically engineered binding proteins as biosensors for fermentation and cell culture
Genetically engineered binding proteins as biosensors for fermentation and cell culture
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DOI：
10.1002/bit.10830
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发表时间：
2003-12-20
影响因子：
3.8
通讯作者：
Rao, G
中科院分区：
文献类型：
--
作者：
Ge, XD;Tolosa, L;Rao, G
对来自大肠杆菌的两种工程化结合蛋白的信号转导特性和潜在应用进行了广泛的研究。这两种蛋白质的多肽链上都有一个半胱氨酸突变，这使得能够引入环境敏感的荧光团：葡萄糖结合蛋白(GBP)的ANS和谷氨酰胺结合蛋白(QBP)的acryloan。这两种蛋白质对其配体的反应都在微摩尔范围内。这些蛋白质可以在4摄氏度的温度下保存至少5个月。表观结合常数、蛋白质浓度和荧光团是影响生物传感器响应范围的三个主要因素。在溶液中，配体的结合是快速和可逆的，但包埋蛋白质的解离平衡和传质阻力不佳会使反应延迟数分钟，恢复延迟数小时。模拟结果表明，使用直径小于或等于1 mm的透析管可以将恢复时间减少到30分钟以内。在150、5和100 mU/L三种不同的规模上，研究了GBP在酵母发酵和大肠杆菌发酵中的潜在应用。测定结果与YSI 2700型化学分析仪进行了比较。虽然后者不能给出可靠的结果，因为在LB培养基中的葡萄糖浓度接近其检测下限，但本文提出的葡萄糖生物传感器成功地应用于每种情况。谷氨酰胺结合蛋白在两种不同规模(100毫升和100毫升)的细胞培养中进行检测，并与YSI法检测结果进行比较。QBP和YSI对100mL的细胞培养都有较好的效果，但YSI的样品量要求较大(至少5mU)，使其不能用于100mL的细胞培养。由于样品体积小(不到1微升)和高灵敏度，这些分析方法在高通量生物处理中可能会得到广泛的应用。(C)威利期刊公司。
The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both proteins respond to their ligands in the micromolar range. The proteins can be stored at 4degreesC for at least 5 months. Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges. The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours. Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes. The potential applications of GBP were studied in yeast fermentation and E. coli fermentations in three different scales: 150 mL, 5 mL, and 100 muL. The results were compared with an YSI 2700 Chemistry Analyzer. Although the latter could not give reliable results for the E. coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation. Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 muL) and the results were also compared with those obtained with YSI. Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 muL) prevented it from being used in the 100-muL cell culture. Because of their small sample volume requirements (less than 1 muL) and high sensitivity, the assays described here might find wide applications in high-throughput bioprocessing. (C) Wiley Periodicals, Inc.