One of the basic characteristics of living cells is the ability to exchange substances and information with the external environment. Through the physiological activities of material and information exchange, cells can sense environmental changes, acquire nutrients necessary for metabolism, and discharge metabolites and waste. The selective absorption and discharge of solutes is mainly achieved by a membrane transport protein-based transport system on the cell membrane.
What are transport proteins?
Transport proteins are a large class of membrane proteins that mediate chemicals and signals exchange inside and outside biofilms. The lipid bilayer forms a hydrophobic barrier around the cell or organelle that isolates it from the surrounding environment. Although some small molecules can penetrate directly through the membrane, most hydrophilic compounds (such as sugars, amino acids, ions, drugs, etc.) require the help of specific transporters to pass through the hydrophobic barrier. Therefore, transporters play an important role in a wide range of cellular activities such as nutrient uptake, release of metabolites, and signal transduction.
The isolation and purification of proteins are the basis for the study of the structure and function of proteins. Under normal circumstances, the expression of most membrane proteins is extremely low, and the bottleneck of membrane protein structure and function research is the lack of effective expression of membrane proteins. Purification techniques are effective means of conducting structural studies. Compared with other types of membrane proteins, membrane transporters are basically single-gene coding products, which can independently perform physiological functions and become a good functional expression and purification research object. The currently used purification methods are: (1) using molecular biology techniques to construct a recombinant membrane protein containing a fusion affinity tag; (2) optimizing the functional expression of the recombinant membrane protein; (3) and isolating and purifying the expressed Membrane protein, and activity testing and structural studies. The advantage of this strategy is that, on the one hand, the introduction of the fusion affinity tag facilitates the detection and purification of the recombinant membrane protein; on the other hand, it is easy to modify and manipulate the target protein, and these modifications and operations such as site-directed mutagenesis can provides important information for the study of protein structure and function.
Construction of recombinant membrane transport protein
When constructing a recombinant membrane protein for in vivo expression, the topological structure of the protein must be considered, especially whether the N-terminus and C-terminus of the expressed membrane protein are located on the cytoplasmic side or the outside of the cell. On the one side, heterologous overexpression of membrane transporter is affected by the N-terminal sequence of the first transmembrane helix located outside the lipid membrane. When constructing a recombinant membrane transporter, whether to introduce a signal sequence-promoting protein based on the position of the N-terminus of the membrane protein expression. In the construction of its expression vector, especially when it is necessary to introduce an affinity tag at the N-terminus, it is necessary to simultaneously introduce and contain a signal sequence in order to successfully express the target protein. Other side, depending on the topological structure of the membrane transporter and the polarity of the amino acid side chain, the type and position of the affinity tag to be introduced can be selected. The affinity tag is an amino acid polypeptide or protein, and an affinity tag is introduced into the recombinant protein. The purpose is to determine whether the target protein is expressed and affinity-purified the target protein. The common feature of the affinity tag is that it can bind to a certain affinity medium, thereby facilitating the affinity purification of the recombinant protein containing the affinity tag. Affinity labeling needs to follow the principle of positively charged amino acid inward, otherwise the expression of the target protein containing the affinity tag will be affected.
Functional overexpression of recombinant membrane proteins
According to the topological properties of membrane proteins, we need to select appropriate expression strategies and optimize expression conditions to obtain a large number of active membrane proteins, which facilitates subsequent purification and structural function studies. Factors that limit membrane protein expression include: lacking of efficient membrane protein folding mechanism or stabilization mechanism in the expression host; degradation of protease; toxicity of recombinant membrane protein to host; inefficiency of protein translation caused by codon preference; post-translational processing of protein Modified or missing modifiers.
Depending on the source of the membrane protein expressed, alternative efficient expression systems include living expression systems such as prokaryotic expression systems and eukaryotic expression systems; and expression systems in vivo. For membrane proteins derived from prokaryotes, the E. coli expression system has achieved good results. The expression and folding of most membrane proteins of Gram-positive bacteria such as E.coli are related to signal recognition particles (SRP). At the same time, inserting into the membrane under the action of SRP completes the folding. The optimization of the functional expression of the recombinant membrane protein aims to control the relative speed of translation and folding to achieve the optimal expression of membrane protein. SRP-mediated membrane protein folding. It also relates to the recognition and folding efficiency of heterologous recombinant membrane protein by the host cell folding mechanism. Therefore, it is necessary to optimize the factors affecting membrane protein translation and folding, for example, the homology of the expression host and heterologous membrane protein source, culture temperature, the composition of the medium, and the choice of co-expressed protein, etc.
Functional expression of membrane proteins derived from eukaryotes often involves a series of processes in protein processing and sorting, and prokaryotic expression systems often fail to achieve satisfactory results. The use of weak protein promoters and the simultaneous expression of molecular chaperones can increase the efficiency of ion channel protein expression. These studies demonstrate that membrane proteins require a certain amount of time and appropriate mechanisms for processing and folding, and ultimately localization, after being synthesized by ribosomes. This is important for the functional expression of membrane proteins. In the living expression system, many factors can lead to the inefficiency of membrane protein expression, for example, the expressed membrane transporter has toxic effects on host cells. In this case, the expression of membrane transporter can be considered by using the in vitro expression system.
In vitro expression systems can also cost-effectively label target proteins for specific studies. The membrane proteins expressed in vitro exist in agglomerated state. After the detergent is added, most of the membrane proteins can be dissolved. The CD structure shows that the secondary structure is mainly α-helix, and the dissolved EmrE, SugE and TehA can be recombined. On the artificial lipid membrane. The transport activity of EmrE recombinant membrane proteoliposome showed that the in vitro expressed EmrE has transport activity. The detergent has two polarities and is stable to the stability of the membrane protein in the membrane-free state. If a suitable detergent is directly added to the in vitro expression system, a membrane transporter present in a dissolved state can be obtained. These membrane proteins can be directly used to construct recombinant membrane proteoliposomes. Studies on MscL recombinant membrane proteoliposomes showed that MscL expressed in vitro after addition of detergent has similar activity to MscL expressed in vitro. Therefore, the membrane transports protein with suitable conformation and functional activity can be obtained by using the in vitro expression system.
Selection of detergents and purification of recombinant membrane proteins
Purification of recombinant membrane proteins containing fusion affinity tags involves three major steps: releasing of membrane proteins from the plasma membrane with a suitable detergent; adsorption of free membrane proteins with affinity chromatography or affinity media; washing away impurities and elution membrane protein. The choice of detergent in this process must consider the following two points: (1) the solubilizing efficiency of the detergent on the target membrane protein; (2) how to remove the detergent after purification for structural and functional studies. Membrane proteins require a plasma membrane to support their structure and function in their natural state, but how to maintain the stability of membrane protein structure and function under the condition of membrane removal during separation and purification is important. Usually, the detergent interacts with the membrane protein and the hydrophobic part of the membrane lipid to weaken the hydrophobic binding between the membrane protein and the membrane lipid molecule, and release the membrane protein from the membrane lipid. The hydrophobic portion of the free membrane protein retains its original conformation under the protection of the detergent, thereby maintaining its original function, such as binding to substrates or inhibitors. Detergents are divided into ionic and nonionic types. Non-ionic detergents and amphoteric detergents have a weaker denaturation effect on membrane proteins, and are commonly used for ion exchange and affinity chromatography of membrane proteins. The solubilizing efficiency of the detergent on the membrane protein is related to the structure of the detergent and the critical micelle concentration (CMC), which can be optimized by changing the type and concentration of the detergent. The appropriate detergent will contain the affinity label. After the recombinant membrane protein is dissolved from the plasma membrane, the affinity membrane is adsorbed by the affinity chromatography column or the affinity medium to carry out the affinity purification experiment. The purification step is similar to the affinity purification step of the soluble protein. However, it must be noted that the concentration of the detergent in the solution must be kept above the critical micelle concentration, while the solution is required to contain high concentrations of glycerol (20%) to maintain the hydrophobic conformation and functional stability of the membrane protein.
In the state of membrane removal, even with the synergistic effect of detergent and glycerin, the stability of most membrane proteins is not strong. Therefore, after purification, membrane proteins must be recombined into artificial lipid membranes as needed to construct membrane protein liposomes, and restore the structure and function of membrane proteins on lipid membranes. In this process, the concentration of detergent should be reduced below its critical micelle concentration, so that the membrane protein is detached from the detergent and inserted into the lipid membrane to form artificial membrane proteoliposome. Methods for removing the scale or reducing the concentration of the detergent include a dilution method, a gel filtration method, a dialysis method, an ion exchange chromatography method, and a special medium absorption method. When using gel filtration and ion exchange chromatography, it is not conducive to the membrane protein interaction with membrane lipid molecules while removing detergent. Dialysis removes detergents at a slower rate and is commonly used for two-dimensional crystallization experiments of membrane proteins. When constructing artificial membrane proteoliposome for functional activity studies, descaling is usually removed by dilution or Biobeads absorption. The use of detergents with high critical micelle concentration facilitates the rapid removal of detergents from the system, so the detergent is exchanged while the membrane protein is affinity purified, and the detergent with low critical micelle concentration is exchanged high. Critical micelle concentration of detergent.