Characterisation of a novel growth hormone variant comprising a thioether link between cys182 and cys189
Characterisation of a novel growth hormone variant comprising a thioether link between cys182 and cys189
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DOI：
10.1002/cmdc.200700042
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发表时间：
2007-08-01
期刊：
影响因子：
3.4
通讯作者：
Jone, Carl
中科院分区：
文献类型：
--
作者：
Datola, Antonio;Richert, Sophie;Jone, Carl
从大肠杆菌生产的生物制药制剂中分离出一种新的重组人生长激素（r-hGH），并对其进行了鉴定和表征。该变体在Cys182和Cys189之间的C端附近含有一个不可约的硫醚桥，并使用各种分析技术进行了表征。正如Cunningham和Wells（7993）先前的工作所强调的那样，这部分序列中的几个残基参与了分子与其受体的结合和亲和力，这种修饰的分子内连接的存在可能对分子的生物学行为具有重要意义。此外，由于治疗性单克隆抗体（Tous et al., 2005）曾报道过将二硫化物转化为硫醚，这可能意味着位于分子这部分的二硫化物桥对硫醚的形成具有普遍的易感性。这反过来又与生物制药工业有关，用于监测蛋白质C末端附近的二硫桥的完整性。目前的研究展示了最先进的物理化学研究，明确阐明了一种新的结构，包括质谱和从头肽测序的肽图谱。近紫外圆二色性和分子模型突出了分子高阶结构的变化。
A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (7993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.