Armisén P, Mateo C, Cortés E, Barredo J L, Salto F, Diez B, Rodés L, García J L, Fernández-Lafuente R, Guisán J M
Hispanagar S.A., Burgos, Spain.
J Chromatogr A. 1999 Jul 2;848(1-2):61-70. doi: 10.1016/s0021-9673(99)00489-6.
A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).
将多聚组氨酸标签融合到克隆于大肠杆菌中的不动杆菌属YS114菌株的戊二酰基酰基转移酶（GA）上，得到了一种具有完全活性的酶。生化分析表明，该标签并未改变嵌合GA（多聚组氨酸GA）的成熟过程，该酶从无活性的单体前体经过复杂的翻译后加工成为有活性的异二聚体酶。这种酶已被用作模型，开发了一种新颖且非常简单的方法，通过在特制载体上进行固定化金属离子亲和色谱一步纯化多聚组氨酸蛋白。其目的是提高目标蛋白在特制螯合载体上的吸附选择性，而非进行选择性解吸。研究了来自大肠杆菌粗提物中的蛋白质以及纯的多聚组氨酸标记的GA在不同金属螯合载体上的吸附速率和程度。高达90%的大肠杆菌蛋白吸附在通过长间隔臂连接到载体上且配体密度高的商业螯合载体上，而当使用低配体密度、短间隔臂以及Zn2+或Co2+作为阳离子时，这种吸附几乎可以忽略不计。相反，多聚组氨酸GA在所有载体上都有足够强的吸附。多聚组氨酸尾与单个螯合部分之间的强亲和相互作用似乎是多聚组氨酸GA吸附的原因。相比之下，涉及多个螯合部分的多点弱相互作用似乎是天然蛋白质吸附的主要原因。通过使用特制的亲和载体，可以进行非常简单的一步纯化GA的操作，同时宿主蛋白的吸附最少。每毫升螯合载体可吸附高达20毫克的GA，而大多数伴随蛋白几乎不吸附在这种载体上。经过几步洗涤后，最终通过用50 mM咪唑洗脱回收目标酶（产率80%），比活性有非常高的提高（高达120倍的纯化倍数）。
