Home Health & Fitness Essential Things to Know About Recombinant Protein Expression 

Essential Things to Know About Recombinant Protein Expression 

One of the organisms of choice for producing recombinant proteins in Escherichia coli. Its use as a cell factory is well established, and it has become the most widely used expression platform. As a result, there are numerous molecular tools and protocols available for high-level heterologous protein production. We review the various approaches for recombinant protein synthesis in E. coli and discuss recent advances in this rapidly expanding field. 

Recombinant Protein Expression   

There is no doubt that recombinant protein expression in microbial systems has transformed biochemistry. The days of needing kilograms of animal and plant tissues or large volumes of biological fluids to purify small amounts of a given protein are almost over. Every researcher who begins a new project that requires a purified protein immediately considers how to obtain it in recombinant form. Additionally, the ability to purify recombinant proteins in large quantities allows for biochemical characterization, industrial process applications, and commercial product development. 

The theoretical steps required to obtain a recombinant protein are pretty straightforward. Have your gene of interest, clone it in whatever expression vector available, transform it into your host of choice, and induce. The protein is ready for purification and characterization.  

Which organism to use?

The host whose protein synthesis machinery will generate the valuable protein will set the tone for the entire process. It specifies the technology needed for the project, which may include a variety of molecular tools, equipment, or reagents. Microorganisms with available host systems include bacteria, yeast, filamentous fungi, and unicellular algae. However, all have advantages and disadvantages, and the protein of interest may influence their selection. 

Significantly, using E. coli as the host organism is well understood: It has unrivaled rapid growth kinetics, and it takes about 20 minutes to double in glucose-salts media under ideal environmental conditions.  

Applications of recombinant protein in research:  

Antibody production   

Animal hosts are inoculated with purified protein multiple times to elicit primary and secondary immune responses. Typically, 13 milligrams of protein are required for antibody production, though the amount varies depending on the animal’s size. The animal’s blood is collected when the IgG immunoglobulin isotype reaches maximum serological levels. However, antibodies can be obtained from samples other than blood (e.g., chicken eggs).  

Aptamer development 

Aptamers, or in vitro nucleic acid-based antibodies, also use recombinant proteins. First, trillions of random RNA or DNA oligos are incubated with the recombinant protein. The protein-binding sequences are then collected and amplified multiple times. Negative selection can also be used during this step to ensure that the aptamer does not bind to proteins similar to the target of interest. Finally, the sequences of aptamers that bind to the target protein are determined, as is the binding affinity of each aptamer. 

Western blot controls 

Western blots are frequently used to determine the presence and amount of a protein of interest in a sample. To ensure that the western blotting procedure is successful, recombinant proteins are used as a positive control. Additionally, recombinant proteins are also used to test the protein’s migration pattern. For more Information https://technewsenglish.com/

ELISA standards  

The enzyme-linked immunosorbent assay (ELISA) is a technique for determining the concentration of a protein of interest. The bicinchoninic acid (BCA) assay is used first to quantify a purified recombinant protein. The recombinant protein is then diluted to generate a standard curve, which is used to calculate the precise protein concentration in the sample. 

Protein interactions 

Recombinant proteins study protein interactions with other proteins, DNA, or small molecules in solution or on a solid substrate. These data could include information about binding partners, binding affinity, or kinetics obtained through immunoprecipitation, isothermal calorimetry, and surface plasmon resonance (SPR). It is also possible to characterize antibody specificity and protein modifications during enzymatic reactions. However, it is critical to consider the expression system and fusion tags of the recombinant proteins for these types of functional assays. 

Protein structure

Protein structure is determined using X-ray crystallography, nuclear magnetic resonance (NMR), and mass spectrometry. An electron map is generated by X-ray crystallography using a minimum of 5 – 10 milligrams of purified, crystallized recombinant protein. However, NMR uses a magnetic field and radio frequencies to measure the distance between atomic nuclei in 2–30 mg of purified protein.  

Cell culture experiments

Cell culture experiments also make use of recombinant proteins. They aid in determining whether a protein can re-establish signaling after a pathway has been blocked. This can clarify why a patient may be resistant to specific targeted therapies. Similarly, recombinant proteins can aid in mapping the relative location of a protein in a signaling pathway. Comparing cellular phenotypes of wild-type and recombinant mutant proteins can help delineate their native function in homeostasis and disease. 


Recombinant proteins are extremely useful in proteomics research. Proteins have two primary advantages over native proteins: greater flexibility regarding which and how many are produced, and purified recombinant proteins are less expensive than purified native proteins.  

Significantly, proteins requiring PTMs should be expressed in PTM-capable systems; large tags may obstruct the binding of epitopes. The large and ever-growing offering of commercially available recombinant proteins reflects the usefulness and versatility of recombinant proteins in research. Furthermore, custom protein services can be used to create recombinant proteins that are not currently available on the market. 

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