Construction of alkali-resistant recombinant protein A based on the EpHod model

• 1、Biotechnology School, Jiangnan University, Key Laboratory of Industrial Biotechnology of Ministry of Education ，Wuxi 214122

Abstract：Protein A (Protein A) affinity chromatography, as the most commonly used antibody purification method, has been explored to improve its longevity and reduce the purification cost. Starting from protein A structural domain Z, we predicted the optimal pH distribution of homologous proteins of protein A structural domain Z monomer and tetramer based on the EpHod model, and screened protein A homologous sequences of basicity, on the basis of which, we predicted the homologous mutation sites using the EpHod model and the MPNN model analysis, and after validation by wet experiments of kinetic simulation, SDS-PAGE and grayscale analysis, we determined that Mu- 29-tetra is largely tolerant to 0.5 M NaOH. 80.6% remains after 24 h immersion in 0.5 M NaOH. The alkaline resistance and reusability of recombinant protein A affinity chromatography media were compared using immunoglobulin G (IgG) antibody protein as the test protein. 90.3% of dynamic load retention was observed after 24 h of immersion in 0.5 M NaOH, and 89.7% of dynamic load retention was observed after simulated reusability for 100 times. The evaluation indexes basically reach the level of commercialized medium, with potential marketable application value.

Keywords： Protein A IgG EpHod alkali resistance modification