The development of next-generation albumin preparation by mutagenesi s studies

人血清白蛋白(HSA)具有维持渗透压、药物结合和抗氧化活性等多种维持体内平衡的重要功能。人血清白蛋白的诱变研究使得研究各种氨基酸残基和结构域参与人血清白蛋白的结合能力、抗氧化活性和延长半衰期等功能性质成为可能。在本研究中，我们首先研究了人血清白蛋白单一残基突变体的抗氧化特性。半胱氨酸残基，特别是^&lt；166和gt；Cys对白蛋白的这一功能贡献很大。其次，利用毕赤酵母分别表达了这三个RHSA结构域。重组结构域I蛋白的抗氧化活性与重组人血清白蛋白相当。设计了一种具有潜在高抗氧化活性的新型结构域交换HSA，将HSA的三个结构域I蛋白基因融合在一起。这种交换的人血清白蛋白具有比野生型人血清白蛋白更高的抗氧化活性。第三，利用巴斯德毕赤酵母表达重组人血清白蛋白二聚体。重组人血清白蛋白二聚体的结构和结合能力与天然人血清白蛋白几乎相同。结果表明，重组人血清白蛋白二聚体具有较好的血管内滞留和较低的血管通透性。最后，单残基突变体R410C只需导入野生型即可。S-NO-R410C具有比S-NO-HSA更高的抗菌活性，本研究结果为进一步开发新型重组白蛋白提供了重要的基础数据。

Human serum albumin(HSA) has many essential functions for homeostasis, such as the maintenance of osmotic pressure, drug binding and antioxidant activity. Mutagenesis studies of HSA made it possible to examine the participation of various amino acid residues and domains in the functional properties of HSA, such as binding capacity, antioxidant activity and prolonged half-life.In this study, firstly, we studied the antioxidant properties of single-residue mutants of human serum albumin. Cysteine residues, especially ^<166>Cys contribute much to this function of albumin. Secondly, Pichia pastoris has been used to express each of the three rHSA domains separately. Recombinant domain I protein exhibited antioxidant activity comparable to that of rHSA. A novel domain exchange HSA with potentially high antioxidant activity was designed where three domain I proteins of HSA were genetically fused. This exchanged HSA had higher antioxidant activity than wild-type HSA. Thirdly, rHSA dimer has been expressed using Pichia pastoris. The structure and binding capacity of rHSA dimer was almost identical to that of native HSA. rHSA dimer was shown to exhibit better intravascular retention and lower vascular permeability properties. Lastly, single-residue mutants R410C efficiently introduced NO further than the wild type. S-NO-R410C had higher antibacterial activity than S-NO-HSA.The results of this research serve as important fundamental data for further development of new recombinant albumin.