Witkowski Andrzej, Krishnamoorthy Aparna, Su Betty, Beckstead Jennifer A, Ryan Robert O
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States.
Protein Expr Purif. 2015 Feb;106:41-8. doi: 10.1016/j.pep.2014.10.015. Epub 2014 Nov 8.
Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects.
Wnt蛋白是一类具有强大生物活性的形态发生素。生物活性重组Wnt的低产量以及分离出的Wnt在没有去污剂的情况下易于自我缔合，阻碍了结构-功能研究。利用稳定转染的果蝇S2细胞，开展了相关研究以提高重组小鼠Wnt3a的回收率，建立无去污剂的Wnt制备条件，并研究有限蛋白酶解的影响。S2细胞培养条件培养基经过三步方案处理，包括染料配体色谱法、固定化金属亲和色谱法和凝胶过滤色谱法。通过选择性合并柱级分，获得了均一且纯化的Wnt3a制剂。用凝血酶对Wnt3a进行有限蛋白酶解导致在N端类鞘脂激活蛋白样基序内发生位点特异性切割。为了生成无去污剂的蛋白质，将Wnt3a固定在带铜（2+）的亚氨基二乙酸衍生化琼脂糖珠上，应用无去污剂缓冲液，并用含有咪唑加30mM甲基-β-环糊精（MβCD）的缓冲液从珠子上洗脱Wnt3a。在含MβCD的缓冲液中回收的Wnt3a是可溶且具有生物活性的。鉴于MβCD是一类能够结合和溶解小疏水配体的无毒低分子量化合物家族的成员，Wnt-环糊精复合物可能有助于在没有不良去污剂影响的情况下进行结构-活性研究。
