Castanheira Pedro, Moreira Susana, Gama Miguel, Faro Carlos
Biocant, Molecular Biotechnology Unit, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, Cantanhede, Portugal.
Protein Expr Purif. 2010 Jun;71(2):195-9. doi: 10.1016/j.pep.2010.02.004. Epub 2010 Feb 10.
Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5-7mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.
拉佛林是一种独特的人类双特异性磷酸酶，因为它含有一个氨基末端碳水化合物结合模块（CBM）。拉佛林基因突变会导致拉福拉病，这是一种进行性肌阵挛性癫痫，会导致早期致命问题。以往生产重组拉佛林的尝试面临各种困难，即出现蛋白质包涵体、被细菌蛋白污染以及蛋白质具有很高的聚集倾向，尽管使用了融合标签来提高溶解度并简化纯化过程。在这项工作中，我们以不含任何融合标签的包涵体形式在大肠杆菌中表达了人类拉佛林。经过快速稀释复性步骤后，通过两步色谱法对蛋白质进行纯化，每升细菌培养物可产生5-7毫克纯化蛋白。纯化后的蛋白显示出具有双特异性磷酸酶的动力学特征以及一个功能性碳水化合物结合模块。通过该方案，我们首次能够生产和纯化不含融合标签的拉佛林，其产量达到传统晶体学结构研究所需的量，为理解拉佛林活性的分子机制以及开发拉福拉病的新疗法铺平了道路。
