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首页> 外文期刊>Biochemistry >Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli.
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Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli.

机译：通过在大肠杆菌中表达来解析人性激素结合球蛋白的类固醇结合和二聚结构域。

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摘要

To determine the minimal sequence requirements for steroid binding and dimerization of human sex hormone-binding globulin (SHBG), the SHBG polypeptide and various SHBG deletion mutants were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Fusion proteins containing the complete SHBG sequence, or the first 177 N-terminal residues of SHBG, bound steroids with high affinity and specificity. Further deletions from the C-terminus severely compromised steroid-binding activity, as did N-terminal deletions beyond residue 18 in the SHBG sequence. Thus, residues 18-177 in SHBG encompass a region required for its steroid-binding activity, and a disulfide bridge normally present between Cys-164 and Cys-188 in SHBG is not obviously essential for steroid binding. Most of the GST/SHBG fusion proteins undergo cleavage at 4 degrees C, releasing immunoreactive polypeptides that correspond approximately in size to their respective SHBG sequences. The 23-kDa immunoreactive cleavage productreleased from the fusion protein containing residues 1-205 in the SHBG sequence (SHBG 1-205) has a 50-fold greater steroid-binding capacity but a 7.5-fold lower affinity than its parent fusion protein. In addition, the 22-kDa immunoreactive polypeptide released from SHBG(1-194) binds steroid, and its dimerization is promoted by steroid ligands that bind SHBG with high affinity. These data suggest that the N-terminal region of SHBG dimerizes readily in the absence of GST and in doing so acquires steroid-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
机译：为了确定类固醇结合和人性激素结合球蛋白（SHBG）二聚化的最低序列要求，在大肠杆菌中将SHBG多肽和各种SHBG缺失突变体表达为谷胱甘肽S-转移酶（GST）融合蛋白。包含完整SHBG序列或SHBG的前177个N末端残基的融合蛋白以高亲和力和特异性结合类固醇。 C末端的进一步缺失严重破坏了类固醇结合活性，SHBG序列中第18位残基以外的N末端缺失也是如此。因此，SHBG中的残基18-177包含其类固醇结合活性所需的区域，并且通常在SHBG中的Cys-164和Cys-188之间存在的二硫键对于类固醇结合显然不是必需的。大多数GST / SHBG融合蛋白在4摄氏度下裂解，释放出大小与它们各自的SHBG序列相对应的免疫反应性多肽。从SHBG序列中包含残基1-205的融合蛋白（SHBG 1-205）中释放的23 kDa免疫反应性裂解产物比其母体融合蛋白的类固醇结合能力高50倍，但亲和力却低7.5倍。此外，从SHBG（1-194）释放的22 kDa免疫反应性多肽与类固醇结合，并且其二聚体通过以高亲和力结合SHBG的类固醇配体促进。这些数据表明，在没有GST的情况下，SHBG的N端区域容易二聚，并因此获得了类固醇结合位点。（摘要截短为250字）

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