BIOLOGY AND MOLECULAR BIOLOGY OF TRANSFORMING GROWTH FACTOR-BETA

在哺乳动物中表达的TGF-β的三种亚型是65-85%同源的 彼此之间。 虽然它们在大多数情况下在功能上是可互换的， 体外试验系统，它们对某些细胞具有独特的活性 例如内皮细胞，其中TGF-β 1和TGF-β 2的活性 3对生长和迁移的抑制作用约为 比TGF-β 2更有效。 试图定义特定的区域 负责这些活性差异TGF-β分子，我们 已经开发了一种表达重组TGF-β的系统， 通过重组DNA方法， 拼接成一个嵌合分子。 迄今为止最有趣的是 是嵌合体，其中成熟TGF-β 1的氨基酸序列的区域 β 1已被拼接到TGF-β 2分子中。 每种重组 TGF-β嵌合体已经使用一系列高纯度纯化至同质。 高压液相色谱步骤。 我们已经证明，中间的三分之一 TGF-β分子的量足以赋予同种型特异性 对内皮细胞的生物活性。 通过制造只含有 选择几个氨基酸的变化，我们希望确定特定的氨基酸 对这种选择性反应负责。 在与此相关的方面， 由于同种型特异性的问题，我们试图描述 TGF-β受体的表达和可能的下游信号传导 中间体，以确定这些是否将共同所有TGF-β 同种型或它们是否将介导同种型特异性作用。

The three isoforms of TGF-Beta expressed in mammals are 65-85% homologous to each other. Although they are functionally interchangeable in most in vitro assay systems, they have distinctive activities on certain cells such as endothelial cells where the activities of TGF-Beta 1 and TGF-Beta 3 on inhibition of growth and migration are approximately 100-fold more potent than that of TGF-Beta 2. To attempt to define specific regions of the TGF-Beta molecule responsible for these differences in activities, we have developed a system for expression of recombinant TGF-Betas engineered by recombinant DNA methodology to have portions of different isoforms spliced into a single chimeric molecule. Most interesting to date have been chimeras in which a region of the amino acid sequence of mature TGF- Beta 1 has been spliced into the TGF-Beta 2 molecule. Each recombinant TGF-Beta chimera has been purified to homogeneity using a series of high pressure liquid chromatography steps. We have shown that the middle third of the TGF-Beta molecule is sufficient to confer isoform-specific biological activity on endothelial cells. By making chimeras with only a few selected amino acid changes, we hope to identify specific amino acids responsible for this selective response. In a related aspect of this problem of isoform specificity, we are attempting to characterize the expression of TGF-Beta receptors and possible down-stream signalling intermediates to determine whether these will be common to all TGF-Beta isoforms or whether they will mediate isoform-specific effects.