Preparation Of Membrane-associated Protein Samples For Solid-state NMR Research

The structure research of membrane-associated protein, a kind of protein arising in physiological or pathological process which is closely related with biomembrane, is very importantto illuminatethe molecular mechanism of biological activities. The structural characterization of membrane proteins has important significance and also is the hotspot domain of recent research.The membrane environment has strongly influence on the membrane-associated protein’s structure and function, so it is necessary to study its structure and dynamics information in the phospholipid bilayer, and it is embodies the advantage of solid-state Nuclear Magnetic Resonance (ssNMR).Both viroporins and β-amyloid peptide have close relationship with biomembrane. Viroporins are a kind of hydrophobic small molecule proteins, which playan important role in virus’s entry, assembling, release, changing the membrane permeability and causing inflammation of host cell.β-amyloid peptide appeared at the pathological process of Alzheimer’s disease. Its deposit on the neurons is considered to be one of the main causes of the disease. Study on its fibrillation and interaction with phospholipid bilayer is of great value for the treatment of the disease.In this research, we combined molecular cloning and protein expression and purification techniques to successfully obtain 3 kind of viroporins, includingSARS-CoV E、PBCV-1 Kcv and ATCV-1 Kcv. The yield of SARS-CoV E finally reached 30 mg per literM9 medium, laying a foundation for solid-state NMR structure research. In this process, we not only screened many factors affecting expression of protein, including fusion tags, inducing conditions, host strains and culture medium, but also finished some work on gene sequence optimization and amino acid sequence optimization from the angle of molecular biology, which provided some reference on the heterologous expression of protein. Moreover, we analyzed and discussed the causes of protein aggregation, which helps to elucidate the molecular mechanism of protein aggregation.In the study of Aβ(1-42), we developed a new method to isolate and purify the protein. Comparing to the previous literature, our method is facile and it also can reduce the protein loss during the period of purification. What is more important is that it is not necessary to heat the samples, so it avoid the adverse effects caused by high temperature (60℃ and 80℃). This method is useful for the purification procedures of other hydrophobic small molecule proteins.