Sainsbury Frank, Jutras Philippe V, Vorster Juan, Goulet Marie-Claire, Michaud Dominique
Département de Phytologie-Centre de Recherche et d'Innovation sur les Végétaux, Université Laval, QuébecQC, Canada; Centre for Biomolecular Engineering, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, BrisbaneQLD, Australia.
Department of Plant Production and Soil Science, Forestry and Agricultural Biotechnology Institute, University of Pretoria Pretoria, South Africa.
Front Plant Sci. 2016 Feb 15;7:141. doi: 10.3389/fpls.2016.00141. eCollection 2016.
The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.
将植物用作重组蛋白的表达宿主，对于生产复杂且具有挑战性的生物制药来说，是一个越来越有吸引力的选择。目前需要一些工具，将用于异源表达的高产基因载体的最新进展与常规蛋白质纯化技术结合起来。在本研究中，我们设计了Cysta标签，这是一种基于蛋白质稳定融合伴侣SlCYS8的新型纯化标签，用于植物表达蛋白的固定化金属亲和色谱（IMAC）。我们表明，Cysta标签可用于在天然条件下轻松纯化蛋白质，然后通过酶促去除以分离目标蛋白。我们还表明，用于连接纯化标签的常用蛋白酶识别位点在常用表达宿主本氏烟草的叶片中稳定性不同，其中易受半胱氨酸蛋白酶作用的接头比丝氨酸蛋白酶可裂解的接头稳定性更低。作为一个例子，我们描述了一种用于一步纯化在本氏烟草中瞬时表达的临床有用蛋白人α1-抗胰蛋白酶的Cysta标签实验方案。鉴于其对基于IMAC的蛋白质纯化市售的各种色谱形式具有潜在适用性，Cysta标签为从植物组织中高效且经济高效地纯化重组蛋白提供了一种便捷方法。
