Saginati M, Siri A, Balza E, Ponassi M, Zardi L
Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Eur J Biochem. 1992 Apr 15;205(2):545-9. doi: 10.1111/j.1432-1033.1992.tb16811.x.
Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.
在此，我们描述了一种从SK - MEL - 28人黑色素瘤细胞系的条件培养基中纯化人腱生蛋白的两步法。第一步是使条件培养基依次通过两个串联的色谱柱。第一个是大容量的明胶 - 琼脂糖亲和色谱柱（用于去除纤连蛋白），第二个是羟基磷灰石色谱柱，第一个柱上未结合的物质直接流经该柱。在这些条件下，条件培养基中存在的所有腱生蛋白都与羟基磷灰石色谱柱结合，然后通过5 - 300 mM磷酸钠梯度将其从该柱上洗脱下来。通过这一步，我们获得了一种粗制的腱生蛋白制剂，相对于起始条件培养基浓缩了约20倍，其中腱生蛋白占总蛋白的50%以上。第二步包括用6%和12.8%的聚乙二醇进行两次连续沉淀。经过这一步后，腱生蛋白的纯度超过95%，并且没有显示出任何已知会与其结合的含硫酸软骨素蛋白聚糖的污染。从21升培养基中我们获得了约3 - 4毫克腱生蛋白，产率约为40 - 50%。该方法具有更高的产率，相对于先前描述的方法更简单，避免了蛋白质暴露于变性剂或苛刻条件下，并且可用于从其他细胞系的条件培养基中纯化腱生蛋白。因此，该方法可能是制备腱生蛋白以研究其生物学功能的一种简单而有用的工具。
