Efficient Expression of Complex Membrane Proteins in Cell-free System and Exploration of Its Industry Application
|Keywords||membrane protein cell-free system liposome aquaporin olfactory receptor biomimetic membrane|
Membrane proteins have been important research targets in protein science, structural biology, pharmaceuticals, and the preparation of biomimetic membrane, due to their significance in biology and medicine. Membrane proteins include both simple membrane proteins with one transmembrane domain and complex membrane proteins with several transmembrane domains. The structure and function researches of complex membrane proteins, including receptors, ion channel proteins, transporters, and enzymes, are involved in various areas of life sciences. However, high-level production of membrane protein in vivo is still challenging, especially for complex membrane proteins. Cell-free protein synthesis system has attracted many attentions as an alternative tool for the production of membrane proteins.The olfactory receptor ODR-10from Caenorhabditis elegans, one member of GPCRs, which are usually targets of drug screening, was investigated in E. coli cell-free system. Both addition of detergents and supplementation of liposomes brought about more than100μg/ml soluble ODR-10. The supplied liposomes could integrate protein synthesis, folding and reconstitution in situ, which will facilitate the structure and function studies and pave the application of biosensors.Besides the large scale production of receptors, the effective expression of channel proteins was investigated. The water channel protein AqpZ was produced in E. coli cell-free system. The protein was effectively expressed and purified by Ni2+affinity chromatography. AqpZ was reconstituted into liposomes successfully and verified to be active. Several natural leader peptides were respectively fused at the N-terminus and verified to enhance the expression level significantly. The supplementation of detergents or liposome could remove the leader peptide in situ, which could simplify the purification process and provide hydrophobic environment for proper folding of active AqpZ. Using this strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system.As we know, membrane proteins can be applied in various industry areas, for example, the preparation of biomimetic membranes. Based on the large production of AqpZ, the water channel chip preparation was explored in this study. The AAO supported lipid bilayer was prepared and amphiphilic diblock copolymer PMOXA-PDMS-PMOXA was synthesized. The interaction of AqpZ with PMOXA-PDMS-PMOXA was investigated. The result indicated that the copolymer was more suitable for preparation of biomimetic membrane incorporated with AqpZ to be applied in seawater desalination or water recycling.Many membrane proteins are attached to sugar chains, and most promising targets in therapeutic application are glycoproteins. How to achieve heterologous glycoproteins is a challenging problem. Thus a simple and effective cell-free system based on yeast was established. Comparing to other eukaryotic cell-free systems, the culturing of yeast cells is low-cost, and the yeast cell-free system is easily prepared. The system can be developed to be suitable for production of glycoproteins which can meet the request of pharmaceutical use by metabolic engineering.This thesis is committed to study the effective production of complex membrane proteins in cell-free system and their applications, as well as the novel cell-free systems investigation for production of various kinds of membrane proteins.