Design Of The Bivalency Binding Peptide For Human Serum Albumin

Human Serum Albumin（HSA）is a natural carrier for drug delivery.In recent years,HSA has been widely used to prolong the half-life of peptide drugs and has made great progress.Our early experimental data,a small peptide（ABD6,LPHSHRAHSLPP）has high affinity binding with HSA obtained by Ph.D.-12 phage display library,with the Kd value of 1.67 μmol/L,and be applied to mediate the release of GLP-1 successfully.The small peptide as a linker between peptide drugs and albumin,this novel sustained-release peptide method can prolong the half-life of peptide drugs.Based on the multivalent molecular thermodynamics,△ G = △ G₁ + △ G₂ → lnK_d1 + lnK_d2 → K_d1 × K_d2,exploring to design a bivalency binding peptide for HSA,which contains the sequences of ABD6-peptide.Inserting a peptide adjacent to the ABD6 binding site to obtain a bivalency peptide could bind with HSA specifically.Two strategies were employed to extend the sequences of ABD6 peptide to obtain a bivalency peptide.First,five random amino acids were extended at the N-terminus of the ABD6 peptide using M13 phage display technology to construct a phage display bivalency peptide library for panning HSA affinity peptide.Second,to prepare the complex crystal of ABD6-peptide and HSA based on structural biology,then to analyze the binding mechanism of ABD6-peptide and HSA by studying the three-dimensional structure of the complex crystal,which provided the structural basis for the reasonable extension the sequences of ABD6 peptide.The construction of phage display bivalency peptide library is divided into two parts.First,the ABD6 peptide sequence is inserted into the phage plasmid M13 KE,and then five random amino acids are introduced.Second,the fusion peptide of ABD6 peptide and 5 random amino acids was inserted into M13 KE.The ABD6-peptide sequence has been inserted into M13 KE correctly.On the other hand,the crystals of HSA were grown in a reservoir containing 150 200 mg HSA,28%³5% PEG3350 and 50 mmol/L potassium phosphate,pH7.5 using hanging or sitting drop methods.HSA crystals were soaked into 9 mmol ABD6-peptide solution for 10 mins,20% glycerol and reservoir used as cryoprotectant,the method lays the foundation for further preparation of complex crystals This study is trying to obtain bivalency peptide with high affinity binding with HSA.The method of peptide drugs binding HSA with non-covalent bond could promote the development of peptide drugs.The bivalency peptide can also be used as affinity ligand to pull down the HSA for large-scale preliminary purification of HSA.