Recombinant Protein Expression
What is recombinant protein? Proteins are one of the most important biological molecules for life. And these proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli and transporting molecules from one location to another. However, when we want to analyze the structure, function, mechanism, pathway or other facts of these proteins, we will find that the quantities are not sufficient from nature source. In that case we need to produce high quantity and purity proteins in a short time and low cost in our lab. Thus, recombinant protein technology is required. Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. Recombinant protein is encoded by recombinant DNA, which has been cloned in a system that supports expression of the gene and translation of mRNA. The recombinant DNA, usually the cDNA sequence of the target protein, is designed to be under the control of a well characterized promoter and express the target protein within the chosen host cell to achieve high-level protein expression. Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein or a large quantity of protein.
Recombinant Protein Expression Systems
E.coli is the preferential one of bacterial systems for recombinant protein production and large scale fermentation as its advantages including fast rate of reproduction, ease of culture, and rich knowledge about its genetics. Bacterial protein expression service offered by Beta Lifescience provides a relatively simple, fast, and inexpensive platform for native and heterologous recombinant protein production.
Yeast protein expression system is a ideally suitable expression system for both secretion and intracellular expression as the yeast offer relatively easy genetic manipulation and rapid growth to high cell densities on inexpensive media. Yeasts are outstanding hosts for the production of functional recombinant proteins with industrial or medical applications. Great attention has been emerged on yeast due to the inherent advantages and new developments in this host cell.The common yeast expression hosts used for recombinant proteins' expression include Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe.
Baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. The baculovirus expression vector system (BEVS) is one of the four major expression systems of genetic engineering. BEVS has several advantages of using the baculovirus-insect cell expression system, such as improved solubility and comprehensive post-translational modifications.
Expression systems utilizing mammalian cells for recombinant proteins are able to introduce proper protein folding, post-translational modifications, and product assembly, which are important for complete biological activity. Mammalian cells are currently the main hosts for commercial production of therapeutic proteins, including monoclonal antibody (mAbs).There are two ways for protein expression of mammalian cells: transient transfection and stable transfection. HEK293 or CHO cells are often used in transient transfection. A small amount of protein can be quickly obtained within one week after the transient transfection.
How to Select the Protein Expression Systems:
Choosing an appropriate protein expression system is the key to the success of recombinant protein expression. Several factors need to be taken into consideration, including target protein property, intended application, protein yield and cost. Furthermore, challenges exist for many protein expression projects, especially for large protein, membrane protein, nuclear protein and proteins with heavy post-translational modifications.
On the right is a brief description of the 4 protein expression systems and their advantages and disadvantages:
Overall, various expression systems have their own advantages and disadvantages.
The use of E. coli expression system is relatively low cost, and can be obtained in a relatively short period of time, but the target protein is often expressed in the form of inclusion bodies, the product purification is difficult, and the post-translational processing and modification system of the prokaryotic expression system is imperfect, and the expression product is less biologically active.
Yeast and insect cell expression systems have high protein expression levels and low cost, but the post-translational processing and modification system is not exactly the same as that of mammals.
Mammalian expression systems produce proteins that are closer to the natural state, but the expression level is low and the operation is cumbersome. Therefore, when choosing an expression system, various factors should be fully considered, such as the nature of the protein to be expressed, the production cost, the expression level, the safety, and the expression cycle.
Beta LifeScience's Custom Protein Production Highlights
- Guarantee: No proteins, No payment.
- One-stop service from gene synthesis to protein expression and purification.
- Vast experience of protein expression.
- Proprietary Protein Expression Platform.
- Multiple protein expression systems: E.coli, yeast, baculovirus-insect and mammalian expression systems.
- High-throughput and large-scale protein expression and production.
- Competitive Pricing and Fast turnaround.
Workflow for Protein Production
The process of protein expression can be divided into several key steps: gene cloning, transfection, screening, expression and purification. First, the target gene is inserted into an expression vector to form a recombinant expression vector. Next, the vector is introduced into the host cell, which is realized by transfection and other techniques. After successful transfection, scientists select cell lines that express the target protein through a screening process. Finally, the target protein with higher purity is obtained through culture and purification steps.
Gene cloning: The target gene is inserted into an expression vector to form a recombinant expression vector. Select the appropriate expression vector, usually choose the expression vector with promoter, activator and selector.
Transfection or transformation: The recombinant expression vector is introduced into the host cell, usually using transfection or transformation techniques. The host cell receives the exogenous gene and starts synthesizing the corresponding mRNA.
Screening: Cell lines expressing the target protein are screened, usually using antibiotics or other markers. The aim is to ensure that only cells with recombinant expression vectors are retained.
Expression: Transcription process: information in DNA is transcribed into mRNA through the process of transcription.
Translation process: mRNA is translated by ribosomes into amino acid sequences to form proteins.
Purification: Collect the cell supernatant by culturing a large number of expressing cells. Purify the target protein by using different protein separation techniques, such as affinity chromatography and ion exchange chromatography.
Validation and analysis: Confirm the purity and activity of the expressed protein. Use techniques such as SDS-PAGE, Western blot, etc. for validation. Perform analysis of structure, function, etc.
FAQs About Our Protein Expression Platforms
There are two common reasons. One is that the label is short, the target peptide chain is relatively large, and the label is not exposed because it is wrapped, we can improve it by adding a linker in the middle of the label and the target fragment; the second is that the structure is incorrect, a high degree of aggregation occurs, and the label is not exposed, the first one is that we can change the construction strategy, and the second one is to optimize buffer so that the label is fully exposed.
We need to deal with the degradation according to the time when it occurs. If the degradation occurs during the culture process, we need to optimize the culture conditions, which may be due to over-abundance of nutrients, etc., and can be improved by optimizing the medium and temperature of culture; if we exclude the degradation occurs during the cultivation process, it may be the degradation occurs during the purification process, and the solution is to add protease inhibitors, treat the environment at a low temperature to reduce the physical shear force.
In order to promote the soluble expression of the protein, you can try to add fusion tags to the N-terminal or C-terminal of the target protein, commonly GST, SUMO tags. However, it is necessary to judge whether tag removal is necessary according to the downstream application of the protein.
Yes, for the E. coli expression system, we are able to produce large quantities of proteins (up to the gram level) in one batch. For these large-scale projects, we usually start with small-scale protein expression and purification, and if the small-scale results meet the customer's needs, we can continue to scale up the experiment. Depending on the customer's needs and protein expression, Beta Lifescience's labs can produce proteins in the milligram to gram scale for both research and industrial customers.
We can learn about the molecular weight of the protein, the presence or absence of signal peptide, transmembrane domains, hydrophilicity, etc., predict the secondary and tertiary structure of the protein from some databases on the Internet, summarize the precautions for protein expression by referring to the literature, and analyze whether to express fusion proteins or not, whether to add tags or not, and carry out codon optimization.
Custom Services
We have rich experience in the expression of recombinant proteins (antibodies, enzymes, membrane proteins, cytokines, etc.) in any species. We provide recombinant protein expression and purification services from small-scale to large-scale.
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