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

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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.