Su Wei Wen, Guan Peizhu, Bugos Robert C
Department of Molecular Biosciences and Bioengineering, University of Hawaii, Agricultural Science 2185N, Honolulu, Hawaii 96844, USA.
Biotechnol Bioeng. 2004 Mar 20;85(6):610-9. doi: 10.1002/bit.10916.
Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.
绿色荧光蛋白（GFP）对于研究植物细胞中的蛋白质运输很有用。这种用途有可能扩展到开发一种高效的分泌报告系统，或实现在线监测植物细胞培养物中分泌型重组蛋白的生产。为此，本研究的目的是：（1）证明并表征转基因植物细胞培养物中荧光GFP的高水平分泌；（2）检验GFP荧光用于监测分泌型重组蛋白生产的效用。在本研究中，我们在烟草细胞培养物中表达了一种分泌型GFP构建体，该构建体是通过将拟南芥碱性几丁质酶信号序列与GFP拼接而成的。培养10至12天后，典型的细胞外GFP积累量为12 mg/L。分泌的GFP具有功能，其占所表达的总GFP的比例高达55%。用布雷菲德菌素A进行培养处理的结果表明，GFP是由培养的烟草细胞通过经典的内质网-高尔基体途径分泌的。在摇瓶培养过程中，培养基荧光随着使用蛋白质免疫印迹法或酶联免疫吸附测定法测定的分泌GFP浓度而增加。通过在线荧光检测对植物细胞培养物中分泌的GFP进行实时监测在生物反应器培养中得到了验证，其中在线培养荧光信号与分泌的GFP浓度呈线性相关。
