Membrane proteins play a key role in basic biological processes. As the main drug targets, membrane proteins have high research value. The production of recombinant membrane protein is the first step in the production of protein tools in biochemistry and biophysics. There are two main difficulties in the production of membrane proteins. One is that the expression of membrane proteins is relatively low. The other is that the hydrophobicity of membrane proteins is very strong, so it is difficult to purify them. To solve these problems, we mainly focus on expression and purification step.
Membrane protein expression.
- Baculovirus-Insect cell expression systems. In order to solve the problem of low expression of membrane proteins in cells, the optimized Baculovirus-insect system was used as the expression cell to express the membrane proteins. Using baculovirus mediated insect cell expression system, we provide a simplified method to promote the production of high-quality virus and the preliminary expression analysis of membrane protein targets. The program steps include virus generation, virus quality control and initial expression test using standard commercial baculovirus vector system. The quality, quantity and expression of recombinant protein were evaluated by SDS-PAGE. In addition, the complete expression level, approximate molecular weight and stability of membrane proteins can be evaluated by small-scale expression and purification experiments.
- Cell-free expression systems. Cell-free platform has achieved both high yields and high-throughput expression of recombinant proteins, The Cell-free platform can produce extracts containing endoplasmic reticulum (ER) vesicles that retained glycosylation and signal sequence processing activity. So, Cell-free platform can be used to produce glycoproteins and membrane proteins. The cell-free system contains eukaryotic folding machinery that can be beneficial for recombinant production of complex eukaryotic proteins. The scalable, low-cost preparation method and the efficient, inexpensive cell-free protein expression method are both developed.
Membrane protein purification.
- Detergent screening. Adding detergent before purification is an effective way to solve the problem of protein hydrophobicity. Surfactants are very important for the operation of membrane proteins. The amphiphilic properties of these components promote the interaction of membrane proteins, which are encapsulated by hydrophobic lipid bilayers in their natural state and become water-soluble. However, solubility cannot be inferred as the stability and recovery of natural functional structures; therefore, although the extraction is good, detergents (surfactants or surfactants) do not have to produce suitable stable membrane protein components. In addition, a detergent that has shown good results previously with a specific membrane protein may not work well with other membrane proteins. In the absence of gold standard or rule of thumb for membrane protein extraction, it is necessary to understand the physical and chemical properties of different detergents before protein extraction. For example, the charge and hydrophobicity of a specific detergent can predict its behavior in solution and the interaction with the selected protein.
- Nanodisc. Nanodiscs are very similar to detergent micelles in structure. It consists of 130-160 lipids, arranged in a bilayer fashion, linked together by amphiphilic proteins called membrane scaffold proteins (MSPs). This gives greater advantages over detergents in terms of stability and solubility. In this method, mild detergents and nanodiscs are used to dissolve the target protein instantaneously. The detergent is removed for further procedure to seal the target membrane protein in the nanodisc assembly. This arrangement mimics a natural state, allowing higher stability at the single molecule level.
For more information regarding Profacgen’s membrane protein expression and purification service, please contact us. Our customer service representatives are available 24 hours a day, Monday through Friday, to assist you.