Design Of Ligands For Human Serum Albumin Separation And Recombinant Human Serum Albumin Purification

Human serum albumin(HS A)has a wide range of uses in clinical treatment.The genetic engineering technology has been used to produce recombinant human serum albumin(rHSA),which is consider to resolve the problem of HSA supply and avoid the risk of blood-borne pathogens.However,the separation of rHSA is a big problem.It is of great importance to develop new high-specific ligands for rHSA separation.In this thesis,Site ? of HSA was selected as the target.Based on the natural ligand L-tryptophan,new ligands were designed by the methods of molecular simulation.New resin was prepared and used for rHSA separating from Pichia pastoris fermentation broth.Firstly,the binding mechanism of Site ? and L-tryptophan was studied by molecular simulation.It was found that the carboxyl and indole groups of L-tryptophan played key roles in the binding.Then seven ligands containing carboxyl and indolyl groups were designed.Molecular simulation results showed that indole-3-valeric acid ligand(DL-5)had the strongest binding ability to HSA.In order to facilitate the preparation of resin,indole-3-acetic acid-cysteine ligand(IAA-CYS)with the structure similar to DL-5 was further chosen.It was found that the binding ability of IAA-CYS ligand to HSA was close to that of DL-5 with molecular simulation.Secondly,new resin was prepared with IAA-CYS ligand.The competitive binding experiment performed with ibuprofen as the competitor.The results indicated that Site ? on HSA would be a potential binding site for IAA-CYS ligand.The molecular model of IAA-CYS-spacer was constructed,and the binding mechanism between IAA-CYS-spacer and HSA was investigated by molecular simulation.It was found that the electrostatic interactions might dominate the binding while the hydrophobic interactions would play an assistant role.In addition,the carboxyl group showed an important role.Finally,the adsorption properties of HSA with IAA-CYS resin were investigated.At the range of pH 4.0-5.0 and NaCl concentration of 0-1.0 M,the adsorption capacity of HSA was higher than 100 mg/mL.It was found that pH 4.5 was the best for HSA adsorption,and the saturation adsorption capacity reached 157.6 mg/mL.The results showed that salt concentration had little effect.When the linear velocity was 100 cm/h,the dynamic binding capacity of HSA reached 59.1 mg/mL.The dynamic binding capacity of HSA was as high as 55.2 mg/mL with the addition of 200 mM NaCl.After the optimization of separation conditions,rHSA was separated from Pichia pastoris fermentation broth.The suitable conditions were loading at pH 4.5,washing with 400 mM NaCl at pH 5.0 and elution with 200 mM NaCl at pH 8.0.The purity of rHSA was 90.1%while the yield was 88.6%.In general,new IAA-CYS ligand was designed and new IAA-CYS resin was prepared for HSA separation.rHSA was separatied from Pichia pastoris fermentation broth with mild loading and elution conditions and high loading amount.The fermentation broth did not need the dilution treatment due to perfect property of salt tolerance.The purity and yield of rHSA separated were high.The results indicated that new IAA-CYS resin had good application potential.