Designing Ligands Of Antibody Affinity Chromatography Based On Molecular Dynamics Simulation And Testing The Binding Ability Of Them

For the past few years,antibody drugs have a lot of social effects, and become an important part of the global pharmaceutical market. In the process of antibody drugs production, the affinity chromatography media used for purification of antibodies cost a lot. And the price of foreign products of antibody affinity chromatography media is high. So cost of antibody drugs production increased a lot.The most widely used affinity ligand is the Staphylococcal protein A(Sp A). It has five highy homologous extracellular antibody-binding regions. All of the 5 domains have the ability of binding the Fc domains and the Fab domains of VH3 family antibodies. The crystal structure formed of Sp A domain D and Ig M Fab segment was analyze and used in the present study as the model. The vital site that inhibits the binding between domain D and Ig M was estimated by means of molecular dynamics(MD) simulation. And we design a mutant of domain D(K46E) for improving affinity with Fab domain. For one thing, there are few studies on Fab binding domains, we can fill in the blanks. For another, the Fab binding ligands can be used for both Ig G and Ig M purification. However, the ligands from domain B can not be used for Ig M purification. The advantage of MD simulation is that we can predict the possible result before the start of the experiment. That will make the experiment more directional.In order to achieve the above research purposes, the thesis is carried out according to the following scheme:1. The MD simulations were performed of the complex structure of domain D and Ig M. We want to find the amino acid sites which hinder the binding betweendomain D and Ig M. And we mutate the sites to get some mutants which have higher antibody affinity.2. Many findings have shown that tetramer ligands have higher binding ability than monomers. So in this study, we expressed the mutant and its tetramer. The expression strain is Escherichia coli BL21(DE3). After fermentation and purification of the ligands, we get the domain D, mutant and its tetramer. And then we compare the antibody(Ig G, Ig M) binding ability of them.3. Compare the binding ability of domain D, mutant and its tetramer with Ig G and Ig M by SPR technology. And get the parameters of ka, kd and KD.4. Coupling the three kinds of ligands to pre-activated medium and verifying the purification capabilities of them with Ig G and Ig M.Through the above experiments, we get the following results:1. After analyzing the structure of wild-type system, it was found that the K46 which contains the positive charge may resist the K2589 and the R2611 in the Ig M.This might be the site that inhibits the binding between domain D and Ig M, thus it was tried to mutate the K46 to reduce such inhibition. Based on the properties of amino acids, it was tried to mutate the K46 to E46. Because E46 contains the negative charge, and does not resist the K2589 and the R2611 in the Ig M. From the comparison of the two minimal systems, we found that the affinity between K46 E and Ig M is stronger than that between domain D and Ig M.2. When we purified domain D, K46 E mutant, and K46 E tetramer, we found that the purification was performed using isoelectric precipitation. The precipitate was re-dissolved using the phosphate buffer(p H 7.5). After this step, the proteins bound Ni column easyer.As for purification of the three proteins, the elution condition for K46 E tetramer was much milder than that for domain D or K46 E mutant, as K46 E tetramer could be eluted under neutral conditions while monomers required a p H value of 3.2.3. Compared to K46 E mutant, domain D and Ig M showed slow binding and slowdissociation, while K46 E mutant showed rapid binding and rapid dissociation with Ig M, displaying significantly increased affinity. That will increase the binding capacity of antibodies. And this could be applied in the development of new affinity media with higher linear flow rate.The association rate, dissociation rate, and affinity of the K46 E tetramer protein with Ig G and Ig M were significantly increased, which is consistent with the previous results(i.e. tetramer increased the affinity of ligands).4. The coupling media of the ligands were used for Ig G and Ig M purification.The results showed that K46 E mutant had higher dynamic binding capacity than domain D in binding Ig G. Unfortunately,in binding Ig M,K46 E mutant did not show higher dynamic binding capacity. The K46 E tetramer coupling medium showed the best binding ability with Ig G and Ig M.In this study, we offer a strategy of designing the ligand of antibody affinity chromatography. The MD simulations were performed of a complex structure. And we mutate the sites which hinder the binding to get some mutants. Then we get the origin protein and mutant protein by expressions, fermentation and purification. The association and dissociation rate constants and affinity of mutants were evaluated at the molecular level using surface plasmon resonance(SPR). Coupling the protein ligands to pre-activated medium and verifying the purification capabilities of them with antibodies. We believe that the strategy of designing the ligand of antibody affinity chromatography can be used in the designing and mutation of other domains in Sp A. And maybe it can be used in designing other kinds of chromatography ligand.