Kusnadi A R, Hood E E, Witcher D R, Howard J A, Nikolov Z L
Department of Food Science and Human Nutrition, Iowa State University, Ames 50011, USA.
Biotechnol Prog. 1998 Jan-Feb;14(1):149-55. doi: 10.1021/bp970138u.
This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.
本研究报道了从转基因玉米种子中生产、纯化和鉴定重组大肠杆菌β-葡萄糖醛酸酶（GUS）和鸡卵清抗生物素蛋白的过程。抗生物素蛋白和gus基因稳定整合到基因组中，并在七代中表达。玉米籽粒中抗生物素蛋白和GUS的积累水平分别为可提取蛋白的5.7%和0.7%。在籽粒内部，抗生物素蛋白和GUS的积累主要定位于胚，这表明泛素启动子可能存在组织偏好性。储存稳定性研究表明，含有GUS或抗生物素蛋白的加工转基因种子可以在10℃下储存至少3个月，在25℃下储存长达2周，而活性不会有显著损失。热稳定性实验表明，整个籽粒中的GUS和抗生物素蛋白在50℃下可稳定保存长达1周。缓冲液组成也会影响从磨碎的籽粒中水性提取抗生物素蛋白和GUS。抗生物素蛋白通过使用2-亚氨基生物素琼脂糖一步纯化，而GUS通过吸附、离子交换、疏水相互作用和尺寸排阻色谱四个步骤纯化。纯化的抗生物素蛋白和GUS的生化特性与各自的天然蛋白质相似。
