Study On Soluble Expression Of Membrane Protein And Its Purification And Analysis

Membrane proteins,the potential targets for many drugs,play a vital role in controlling material transport,signal transfer and energy exchange.As a result,it is of great importance to understand their structure accurately.However,due to the low abundance and strong hydrophobicity of membrane proteins,researchers may find it difficult to obtain enough samples and they have to dissolve membrane proteins into aqueous phase with the help of surfactants.Here,in this article,we studied the effects of different hydrophilic tags and their tag positions on the expression of membrane proteins by means of molecular biology methods based on three different membrane proteins called AqpZ,EmrE and MscL.We also investigated the effects of amphiphilic protein ApoAI fused to the C-terminal of the proteins on the solubility of membrane proteins.We would determine the best conditions for the expression of fusion protein,purify fusion protein by means of affinity chromatography and size exclusion chromatography and analyze protein samples by native mass spectrometry.The results showed that C-or N-terminal fused TrxA tag could improve the expression level of membrane proteins to a great extent.ApoAI of C-terminal fusion can realize the solubilization of membrane proteins.We got enough protein samples which possess good mass spectra signal through native mass spectrometry analysis with their purity over 99%after the purification in the best expression condition.By using the method of fusion expression,we overcame the problem of low abundance in the process of membrane protein expression and reduced the difficulty of purification without surfactants.The analysis is much simpler and provides new ideas for studying membrane protein structures and functions and for finding more potential drug targets in future.