Improvement of protein expression by Escherichia coli based on the structure of anti-P2X4 antibody Fab and development of its additional function.

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K06517
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
• Research Institution: International University of Health and Welfare
• Principal Investigator: Abe Yoshito 国際医療福祉大学, 福岡薬学部, 教授 (60315091)
• Co-Investigator(Kenkyū-buntansha): 山下 智大 九州大学, 薬学研究院, 講師 (30645635)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: 抗P2X4抗体 / タンパク質工学 / タンパク質化学 / 大腸菌発現

Outline of Final Research Achievements

We established a method to prepare soluble anti-P2X4 Fab from Escherichia coli without using a time-consuming and laborious refolding system, and found that the amount of soluble Fab recovered increased 6-7-fold in H-S35AFab, where the H-chain Ser35 was mutated to alanine, without altering binding affinity. In this study, we investigated the cause of the increased expression of H-S35AFab by mutating Ser35 in the H-chain, as well as the changes in structure and stability. As a result, we discovered that the expression level of antibody Fab depends on the hydrophobicity of the 35th amino acid side chain of the H-chain, and that stability and expression level are not generally proportional. Additionally, we attempted to prepare functional antibodies by chemically cross-linking mutants of antibody Fab with cysteine and ATP-hydrolysing enzymes, and by adding sugar chains or polyethylene glycols to antibodies to inhibit aggregation.

Free Research Field

タンパク質科学

Academic Significance and Societal Importance of the Research Achievements

低分子化抗体Fabは安定性、抗原親和性が優れており、抗体医薬品の次世代フォーマットとして期待されている。Fabは大腸菌発現から得られたリコンビナントが医薬品にも使われているが、リコンビナントFabは各抗体依存的に発現量が少ないものも多い。よってアミノ酸変異を用いて、大腸菌発現量を理論的にコントロールできれば抗体製剤の発展に繋がっていく可能性があると考え、本研究を行った。