Study On Protein Immobilization Method Based On Tyrosine Chelating Capture And Photo Induced Crosslink

The modification and immobilization of protein is an important supporting technology for its application in the fields of bio-separation,immobilized enzyme catalysis,analysis and diagnosis.The ideal immobilization process depends on the coupling reaction with mild conditions,high efficiency and high utilization.However,the covalent coupling of proteins on the surface of solid-phase carrier materials is a two-phase reaction,and there are typical characteristics of steric hindrance and low active site concentration,resulting in widespread problems of low reaction efficiency and low utilization of active molecules.Aiming at the common problems mentioned above,this study proposes a protein immobilization strategy based on a two-step method which consists of chelating capture and coupling reaction.The research idea is to modify the tyrosine on the surface of the solid-phase medium through amino group,use the carboxyl group in tyrosine to chelate metal ion to achieve the adsorption and capture of the histidine tag fusion protein,and then achieve the photocatalytic coupling of the phenol group in captured protein molecules.This technical strategy will effectively improve the mass transfer effect and increase the utilization rate of protein.The specific research contents are as follows:(1)Preparation of tyrosine-modified carrier medium and its adsorption of histidine-tagged protein.The agarose was coupled with tyrosine by the bromo propylene activation method to prepare agarose tyrosine coupling density of 25?mol/m L.Studies have found that agarose gel coupled with tyrosine can chelate nickel ions,thereby effectively capturing histidine tag fusion proteins.Using green fluorescent protein as the model system,the saturated adsorption capacity can reach 65 mg/m L,and 200 m M imidazole can elute about 95%of the bound protein,which proves that tyrosine has a function similar to NTA and other metal chelating ligands.(2)Optimization of reaction conditions for chelation enrichment-photocatalytic immobilization of protein.Based on the immobilization rate and activity retention rate of green fluorescent protein,the in-situ photo-crosslinking immobilization process of chelated adsorbed protein was optimized.The results show that the chelation enrichment effect can effectively solve the self-crosslinking of protein in the photocatalytic immobilization reaction.The problem has significantly improved protein utilization.Illumination time and catalyst dosage have a significant effect on protein immobilization rate and activity retention.The optimal reaction conditions are 5-10?L catalyst dosage(0.05 M Ru²⁺,1 M AP)and light for 5 min.Compared with the traditional primary amino group immobilization method,the chelation enrichment-photocatalytic immobilization method can increase the utilization rate of active protein by about 20%.On this basis,the protein immobilization technology was successfully applied to the immobilization of recombinant Staphylococcus aureus protein A and Nanobodies.In this study,using the characteristics of tyrosine with multiple functional groups,a two-step"chelation-immobilization"covalent coupling method was established,which can significantly increase the utilization rate of the active protein to be immobilized and improve the immobilization process.The economics of the process have potential application value in the fields of biological separation and analysis.