OBJECTIVES The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein. METHODS Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms. RESULTS Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an alpha-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained. CONCLUSIONS The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.

中文翻译：

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5 kDa生长激素同工型的表征。

目的人类生长激素（GH）的43个氨基酸的5 kDa N端片段显示出特异性和显着的体内胰岛素样活性。该同种型可以通过固相合成方法容易地获得。我们在这项研究中的目的是详细描述此过程，并提供蛋白质的结构信息。方法采用固相合成法合成5 kDa GH同工型。已经进行了圆二色性和有限的蛋白水解作用以提供有关蛋白质在溶液中的折叠状态的结构信息。使用表面等离振子共振在抗体结合水平上比较合成蛋白和蛋白水解同源物之间的结构等效性。以此目的，产生了一种对5 kDa同种型具有特异性的鼠单克隆抗体，并利用该蛋白和其他几种GH同种型对其进行了表征。结果圆二色性和蛋白水解结果表明5 kDa蛋白的C末端片段在α螺旋中折叠。在抗体结合水平上将合成产物与其蛋白水解同源物进行比较表明了结构等效性。产生了针对5 kDa GH同工型的高度特异性抗体，对17、20和22 kDa同工型没有交叉反应。获得了与合成的5kDa GH相互作用的动力学数据。结论与包含在22 kDa GH同工型中的蛋白质相比，该蛋白质的结构似乎有所不同。最后，已经产生了高度特异性的抗体。