Identification Of Recombinant Pichia Pastoris Expressing FcγRI-albumin Fusion Protein And Characterization And Functional Study Of The Target Protein

Bispecific antibodies have become a research and development hotspot in the field of antibody drugs because they can recognize two antigenic epitopes and greatly increase the titer of the antibody.However,its structural design and preparation process are much more complex than monoclonal antibodies,and its production and application are limited to a certain extent.In the early stage,our research group developed a universal antibody immobilization platform(called "nano-adaptor")by bonding anti-Ig G Fc antibody(αFc)to aminated polystyrene nanoparticles.The system mounts the immobilized antibody through the interaction between the Fab segment of αFc and the conserved Fc domain of the monoclonal antibody drug.Such nano-adaptor technology realizes the convenient and controllable preparation of bispecific antibodies and endows monoclonal antibody drugs with high efficiency.However,the process of bonding anti-Fc antibodies to the surface of nanoparticles is complicated and the quality control is difficult.This project proposes to replace αFc with a fusion protein(FcγRI-SA)recombined with Fcγ receptor I/CD64(FcγRI)and serum albumin(SA),which can form a fusion protein complex nano-adaptor with polylactic acid.In this study,the recombinant Pichia pastoris engineered bacteria expressing FcγRI-SA fusion protein were detected and identified,the fermentation conditions of the target protein were optimized,and the target protein was characterized and functionally studied.This project firstly constructed a seed bank of engineered bacteria expressing mouse FcγRI(Mouse Fcγ receptor I,m FcγRI)-serum albumin(Mouse serum albumin,MSA)fusion protein,and tested its growth characteristics.The growth state,cell morphology,phenotype,integration of target genes,etc.of the main seeds and working seeds under different medium cultivation conditions confirmed that the engineered bacteria expressing the m FcγRI-MSA fusion protein had the growth and reproduction characteristics of Pichia spp.More than 95%of the seed batches are integrated with target gene fragments;by optimizing the fermentation conditions of the engineered yeast,a scheme for efficient and stable production of the target protein has been optimized;three batches of m FcγRI-MSA fusion proteins were used as the research objects,and SDS-polyacrylamide gel electrophoresis(SDS-PAGE),matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),ultraviolet and visible spectrophotometry(UV-Vis),capillary isoelectric focusing-whole column imaging detection(c IEF-WCID),high performance liquid chromatography(HPLC)and other methods were used to identify tlhe basic physical and chemical properties,primary structural sequence and impurity residues of the target protein;we proved that m FcγRI-MSA binds to the monoclonal antibody with high drug efficacy;at the same time,we also proved that the constructed fusion protein complex nano-adaptors which based on m FcγRI-MSA and biomedical polymer materials can be used for construction of bispecific antibodies.This study preliminarily proves that FcγRI-albumin fusion protein can be used to construct a universal antibody immobilization platform,which lays the foundation for preclinical testing and drug development of nano-adaptors.