Ford C F, Suominen I, Glatz C E
Department of Food Science and Human Nutrition, Iowa State Uiversity, Ames, 50011.
Protein Expr Purif. 1991 Apr-Jun;2(2-3):95-107. doi: 10.1016/1046-5928(91)90057-p.
Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.
已经开发了几种融合尾系统，以促进从粗细胞提取物或培养基中高效回收和纯化重组蛋白。在这些系统中，目标蛋白经过基因工程改造，含有C端或N端多肽尾，这为回收和纯化的特异性提供了生化基础。已经使用了具有各种特性的尾：（1）对固定化底物或抑制剂具有亲和力的完整酶；（2）对免疫球蛋白G或白蛋白具有亲和力的肽结合蛋白；（3）碳水化合物结合蛋白或结构域；（4）用于体内生物素化的生物素结合结构域，可促进融合蛋白与抗生物素蛋白或链霉抗生物素蛋白的亲和力；（5）对固定化单克隆抗体具有亲和力的抗原表位；（6）用于基于电荷的回收方法的带电荷氨基酸；（7）用于通过固定化金属亲和色谱法回收的聚（组氨酸）残基；以及（8）其他聚（氨基酸），其结合特异性基于氨基酸侧链的性质。融合尾在实验室规模上很有用，并且有可能使用经济的回收方法提高回收率，这些方法易于扩大规模用于工业下游加工。融合尾可用于促进目标蛋白的分泌，还可基于酶活性或抗体结合提供有用的检测标签。许多融合尾不会干扰目标蛋白的生物活性，在某些情况下还被证明能使其稳定。然而，为了纯化天然蛋白，通常会包含一个特异性切割位点，以便在回收后去除尾巴。
