Kusnadi A R, Evangelista R L, Hood E E, Howard J A, Nikolov Z L
Department of Food Science and Human Nutrition, 2312 Food Science Building, Iowa State University, Ames, Iowa 50011, USA.
Biotechnol Bioeng. 1998 Oct 5;60(1):44-52. doi: 10.1002/(sici)1097-0290(19981005)60:1<44::aid-bit5>3.0.co;2-0.
The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.
为改善农艺性状而开发的植物生物技术工具，如今正被用于为食品、饲料和制药行业生产重组蛋白产品。本研究探讨了与利用转基因玉米作为蛋白生产系统相关的几个加工和蛋白回收问题。编码β-葡萄糖醛酸酶（rGUS）的gus基因在四代中稳定整合并表达。rGUS的积累水平达到可提取总蛋白的0.4%。在籽粒中，尽管使用组成型泛素启动子指导gus表达，但rGUS仍优先在胚中积累。利用第四代转基因种子研究种子加工对rGUS活性和回收率的影响。含有rGUS的转基因种子可在室温下储存两周，在10℃下储存至少三个月，酶活性无显著损失。暴露于干热的rGUS在磨碎的种子中比在完整籽粒中更稳定。酶稳定性与加热过程中样品的水分损失相关。对转基因种子进行干磨、分级和己烷萃取，以生产全脂和脱脂胚部分。从磨碎的籽粒、全脂胚和脱脂胚样品中进行水相萃取rGUS的结果表明，每克固体中从磨碎的全脂胚和脱脂胚中萃取的rGUS比从籽粒样品中多约10倍。用热己烷（60℃）从磨碎的胚中萃取玉米油不影响可萃取的rGUS活性。通过离子交换、疏水相互作用和尺寸排阻色谱从磨碎的籽粒和全脂胚提取物中纯化rGUS。两种提取物获得了相似纯度和产量的rGUS。从转基因玉米种子中纯化的rGUS的生化特性与大肠杆菌GUS相似。
