Protein expression has attracted attention in biochemistry since it has become a powerful laboratory technology in the field. Protein expression can be defined as how living cells produce and modify proteins. Custom protein expression can also be referred to as laboratory technology to synthesize protein. The genes are encoded through a process known as gene expression, which is modified to produce a particular set of proteins through protein expression.
The protein expression system uses a defined process that adequately ensures the production of pure protein. The following article will take you through five major things you need to know about custom protein expression.
Strong promoter has to be regulated.
The promoter has a significant influence on the whole process of protein expression. The strong promoters can be dangerous to the cells and hence have to be regulated. Unregulated promoters can lead to loss of the plasmid, which is responsible for hosting the promoter, or even leads to the constitutive synthesis of the promoter, which is detrimental to the host cells. The regulation of the promoter involves turning it on at a specific stage in the growth of the host cells and hence should not always be turned on in the whole process. To achieve this, you will be required to add a specific metabolite or changes in the temperature of the growth medium.
Regulating the strength of the promoter will ensure that the expression of a foreign gene does not interfere with normal cellular gene function and has no adverse effects on the cells. When you entrust your manufactured or synthetic antibody manufacturing to professional antibody production services, you have the extra benefit of faster turnaround times. To regulate this promoter, you have to add 3-indoacrylic acid, which will prevent distortion of the host cell.
Foreign Gene expression effects
Genes are responsible for encoding protein. Therefore, gene expression is a crucial factor in protein expression as it determines the functions of a particular cell. Foreign genes expression is affected by a variety of factors such as the transcriptional genes, the ribosome binding sites, the location of the coned protein within the host cell, the metabolic rate of the cell, the stability of the codons utilized in the foreign gen and the number of foreign genes expressed. The number of particular proteins synthesized in any cell depends on the balance between the protein's synthetic and degradative biochemical pathway.
Therefore, there should be a regulation of genes expression through the whole process of protein synthesis. Gene expression is regulated by introducing the nucleus, especially at the transcriptional level controlling the eukaryotes. Notably, only a specific number of genes are expressed in cells at a particular time. Gene expression is usually affected by environmental changes and requires some regulatory precautions. The availability of nutrients tends to affect the prokaryotes genes. The nutrients allow the bacterial organisms to change the transcription patterns in response to environmental conditions effectively.
Protein purification
Protein purification is a combination of procedures for isolating proteins from a complicated mixture, which frequently consists of cells, tissues, or complete organisms. Purifying the protein of interest is critical for determining its function, interactions, and structure. Purification can also separate the mixture's protein and components, dividing required proteins from every other protein. The time-consuming component of protein purification is typically separating a single protein from the others.
Differences in Physico-chemical characteristics, protein size, biological activity, and binding affinity are commonly used in separation procedures. Protein isolate is one of the words for the pure outcome. Protein purification is a significant stage in custom protein expression that can be done preoperatively or analytically.
Protein expression host
The host cell will affect the process and the final output during protein expression. The central protein expression host includes; mammalian systems, yeast, and bacterial system. Most technicians doing custom protein expression prefer using bacterial expression host as they are aggressive and straightforward, especially in expressing the recombinant protein. The bacterial expression host has a short doubling time, making them convenient as the expression host cells.
Most technicians widely use E. coli bacterial host cells as adequate information about their genetics and physiology. Further, E. coli can easily be manipulated, grow to high densities, and is convenient for large-scale protein expression. However, it's good to note that bacterial host systems contain toxic pyrogens, and hence the synthesized protein should be effectively tested. Therefore, when choosing a protein expression host, you must consider a suitable purification method.
Removal of endotoxin
Removing endotoxin is one part of protein expression. The endotoxins are majorly attached to the outer cell membrane of the host cell. The outer cell wall membrane always consists of components that can lead to inflammatory effects on the host cells, leading to lethal damage. Therefore, during the custom protein expression process, removing these endotoxins is essential, especially in the final stage of the protein preparation.
Different endotoxin removal methods include; monocyte activation test, recombinant factor c assay, and Limulus amebocyte lysate test. The endotoxin in protein can be toxic and lethal, and hence this is a crucial stage in custom protein expression.
Final thoughts
Protein expression is a process by which living things synthesize proteins. This process by a living organism has attracted attention from most scientists to synthesize and modify protein through laboratory technology organically.