DataSheet1_Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.docx

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

人类胎儿血红蛋白（HbF）是开发氧治疗剂的极具潜力的候选起始蛋白。这要求HbF能够在异源表达系统中实现高水平、均一化的制备。在HbF的α链表面引入额外负电荷，可提升功能性蛋白在大肠杆菌（Escherichia coli）中的重组表达产量。本研究针对一种在每条α链上携带四个额外负电荷的HbF突变体（rHbFα4）的结构、生物物理及生物学特性开展了表征分析。本研究通过X射线晶体学以1.6埃的分辨率解析了rHbFα4突变体的三维结构。除可提升大肠杆菌中重组蛋白的表达产量外，本研究还发现rHbFα4突变体的正常DNA切割活性显著降低，其速率常数较野生型降至原有的四分之一。rHbFα4突变体的氧结合特性与野生型HbF完全一致。在本次研究考察的氧化速率（包括自氧化及过氧化氢介导的高铁血红素（ferryl）形成过程）方面，野生型HbF与rHbFα4未表现出显著差异。但高铁血红素还原反应则存在一定差异，该差异似乎与α链相关的反应速率有关。