State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China.
Biochimie. 2012 Apr;94(4):1001-8. doi: 10.1016/j.biochi.2011.12.022. Epub 2012 Jan 10.
A variety of tumour necrosis factor α (TNF-α) derivatives have been bioengineered to improve antitumour activity and reduce toxicity. The expression of TNF-α in Escherichia coli usually yields a mixture of homotrimers and monomers; however, only the trimer shows antitumour activity. TNF-αD10, a bioengineered hTNF-α derivative, demonstrated 10-fold higher cytotoxicity against tumour cells compared to hTNF-α, but the trimer to monomer ratio was 58:42. In the present study, we investigated the structural differences between the trimer and the monomer of TNF-αD10. We found that the chemical shifts of the C-terminal Trp(114) in the trimer were significantly different from those in the monomer and that the replacement of Trp(114) with different amino acids remarkably reduced the trimer production. Further analysis of the publicly available X-ray crystallographic data for trimeric and monomeric hTNF-α revealed that the conformation of the U-shaped region formed by the fragment Cys(101)-Trp(114) was different between the two forms: a hydrophilic cavity in the monomer and a hydrophobic cavity in the trimer. These findings suggested the potential approaches of molecular and structural modification for future improvement of hTNF-α trimer production.
已经对多种肿瘤坏死因子 α（TNF-α）衍生物进行了生物工程改造，以提高抗肿瘤活性并降低毒性。大肠杆菌中 TNF-α 的表达通常会产生三聚体和单体的混合物；然而，只有三聚体具有抗肿瘤活性。TNF-αD10 是一种经过生物工程改造的 hTNF-α 衍生物，与 hTNF-α 相比，对肿瘤细胞的细胞毒性高 10 倍，但三聚体与单体的比例为 58:42。在本研究中，我们研究了 TNF-αD10 三聚体和单体之间的结构差异。我们发现三聚体中 C 末端色氨酸（Trp114）的化学位移与单体中的明显不同，并且用不同的氨基酸替换色氨酸（Trp114）会显著降低三聚体的产量。对公开的三聚体和单体 hTNF-α 的 X 射线晶体结构数据的进一步分析表明，由残基半胱氨酸（Cys101）-色氨酸（Trp114）组成的 U 形区域的构象在两种形式之间有所不同：单体中是亲水腔，三聚体中是疏水腔。这些发现表明了未来提高 hTNF-α 三聚体产量的分子和结构修饰的潜在方法。
