Recombinant protein expression technology helps investigate gene regulation and protein structure-function. Recombinant protein expression has many uses, from in vivo functional studies to large-scale manufacturing for structural studies and biotherapeutic medicine discovery.
This handbook will cover the principles of protein expression, from selecting a host system to building your protein expression vector, and emphasize crucial ideas and items for optimizing recombinant protein output and purification.
Which Organism to Use?
The host cell whose protein synthesis machinery will produce the functional protein and will set the tone for the entire procedure. Therefore, it specifies the technology required for the project, which could include several molecular instruments, equipment, or reagents.
Microorganisms with suitable host systems include bacteria, yeast, filamentous fungus, and unicellular algae. However, each has advantages and disadvantages, and the protein of interest may influence their selection. In this review, we will focus specifically on Escherichia coli.
The benefits of utilizing E. coli as the host organism are widely understood. They include:
- It exhibits unrivaled rapid growth kinetics.
- High cell density cultures are simple to create.
- Rich, complex media can be made using easily available and low-cost components.
- Exogenous DNA transformation is quick and straightforward.
Which Plasmid Should Be Chosen?
Today's most frequent expression plasmids result from different combinations of replicons, promoters, selection markers, multiple cloning sites, and fusion protein/fusion protein removal techniques.
As a result, the catalog of accessible expression vectors is vast, and it is simple to become befuddled when deciding which one to use. These qualities must be carefully analyzed based on the particular demands to make an informed decision.
Transformation and plasmid isolation
Introducing exogenous DNA into the host cell is known as plasmid or vector transformation. Transformation usually refers to incorporating DNA into bacterial, yeast, or plant cells, whereas transfection is reserved for mammalian cells. Chemical transformation, electroporation, or particle bombardment are common methods for transforming a DNA construct into a host cell.
Chemical transformation makes cells competent (capable of taking in foreign DNA) by treating them with divalent cations such as calcium chloride, making the bacterial cell wall more permeable to DNA.
Heat shock creates temporary breaches in the cell membrane, allowing exogenous DNA to enter the cell. However, a brief electrical pulse is utilized in electroporation to permeate the bacterial cell momentarily. Particle bombardment is commonly employed to change plant cells. The DNA construct is coated on gold or tungsten particles, which are then physically driven into the cell via a gene gun.
Several factors influence plasmid transformation success, including antibiotic concentration, construct size and concentration, and ligation efficiency. Plasmids are transformed into competent cells for propagation and storage.
It is critical to consider the following aspects when selecting a competent cell strain to work with: Genotype, transformation efficiency, application, and kit format are all factors to consider.
Selecting an expression system
The key to success is to use the correct expression; each system has its advantages and disadvantages that must be considered while selecting an expression system. However, there may be a need to have a custom protein expression system for your application in case of any technicalities. Protein solubility, functionality, purification speed, and yield are critical when selecting an expression system.
Once a system has been chosen, the gene delivery technique for protein expression must be considered. Transfection and transduction are the two most common methods for delivering genes.
Transfection is the method of introducing nucleic acids into human and insect cells. Protocols and techniques vary greatly, but some examples include lipid transfection and chemical and physical approaches such as electroporation.
Viral vectors are frequently used to transfect cell types that are not susceptible to lipid-mediated transfection. Virus-mediated transfection, also known as transduction, is the most often employed approach in clinical research for reaching difficult-to-transfect cell types for protein overexpression or knockdown.
Cell lysis and protein purification
As the purpose of cell lysis is to purify or evaluate the function of a specific protein, significant consideration must be given to the impact of the lysis reagents on the protein(s) of interest's stability and function. Certain detergents will inactivate the function of specific enzymes, and the long-term stability of extracted/purified proteins frequently necessitates their removal from the original lysis reagents and stabilization with specific chemicals.
Protein yield and activity can be increased by using the proper lysis reagents and purification resin. Additionally, cell lysis formulations are available for specific host systems, such as a cultured mammalian, yeast, baculovirus-infected insect, and bacterial cells.
Bottomline
E. coli has historically been the chosen microbial cell factory for recombinant expression. E. coli is an effective host for expressing globular proteins. The procedures and tactics for protein expression and purification have often been described in good, comprehensive ways for the professional.
