Joh Lawrence D, McDonald Karen A, VanderGheynst Jean S
Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
Biotechnol Prog. 2006 May-Jun;22(3):723-30. doi: 10.1021/bp0503235.
Extraction and storage of a recombinant protein produced by transient expression following agroinfiltration of lettuce were investigated. Lettuce leaves expressing beta-glucuronidase (GUS) were extracted by homogenization in several buffer combinations, and the yield and stability were assessed. The reducing agent dithiothreitol (DTT) was found to be the most important (significant) component in the extraction buffer. An extraction buffer consisting of 50 mM sodium phosphate at pH 7.0 with 10 mM DTT produced a good yield and stabilized GUS. Leaching of GUS through intact agroinfiltrated lettuce leaves was determined to be infeasible, with a maximum flux of 10 microg GUS/h/m2 and recovery of 1.7% of the GUS content in 24 h. Freeze-drying was evaluated as a method to extend the shelf life of the perishable leaf material containing GUS. First- and second-order kinetic models and the Weibull distribution were compared to describe inactivation of GUS in the freeze-dried leaves and leaf extracts. The first-order model best fit the inactivation data. An Arrhenius model fit the first-order inactivation data with respect to temperature with R2 = 1.00. Freeze-drying the lettuce leaves extended the estimated half-life of GUS to 69 days at 21 degrees C versus 11 days at 4 degrees C for fresh lettuce.
研究了通过农杆菌浸润生菜后瞬时表达产生的重组蛋白的提取和储存方法。用几种缓冲液组合对表达β-葡萄糖醛酸酶（GUS）的生菜叶片进行匀浆提取，并评估产量和稳定性。发现还原剂二硫苏糖醇（DTT）是提取缓冲液中最重要（显著）的成分。由pH 7.0的50 mM磷酸钠和10 mM DTT组成的提取缓冲液产生了良好的产量并使GUS稳定。经测定，GUS通过完整的农杆菌浸润生菜叶片浸出是不可行的，最大通量为10 μg GUS/小时/平方米，24小时内GUS含量的回收率为1.7%。评估了冷冻干燥作为延长含GUS的易腐叶片材料保质期的方法。比较了一级和二级动力学模型以及威布尔分布，以描述冷冻干燥叶片和叶片提取物中GUS的失活情况。一级模型最适合失活数据。阿伦尼乌斯模型拟合了关于温度一级失活数据，R2 = 1.00。冷冻干燥生菜叶片将GUS的估计半衰期延长至21℃时为69天，而新鲜生菜在4℃时为11天。
