Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Headington, OX3 9DS Oxford, UK.
BMC Biotechnol. 2013 Sep 24;13:74. doi: 10.1186/1472-6750-13-74.
The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting.
Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein.
Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.
谷氨酰胺合成酶的蛋白表达系统广泛应用于工业和学术界，用于生产重组蛋白，但依赖于转染细胞的克隆，这需要大量的时间和处理。我们通过共表达内部核糖体进入位点的绿色荧光蛋白，并使用荧光激活细胞分选选择高绿色荧光蛋白表达细胞，简化了该系统的中国仓鼠卵巢细胞蛋白产生培养物的生产。
虽然其他利用绿色荧光蛋白和荧光激活细胞分选选择的表达系统依赖于两个或更多的分选步骤，但我们在单个分选步骤后，获得了测试蛋白的稳定表达，其水平> 50%的“平均”克隆和~40%的“最佳”克隆。与基于克隆的选择相比，主要的节省在于处理步骤的数量（减少三分之一）、处理时间（减少 70%）和产生蛋白表达培养物所需的时间（减少约 3 周）。通过这种方式将谷氨酰胺合成酶的表达系统与产品无关的选择相结合，也促进了难以检测的蛋白的生产。
通过仅使用单个基于荧光激活细胞分选的选择步骤，新的简化谷氨酰胺合成酶蛋白表达系统的实施提供了足够用于大多数研究目的的蛋白产量，其中<10mg/L 的蛋白表达通常就足够了，但通常需要多次试验相对大量的构建体。
