Researchers can now express heterologous proteins in various biological systems due to advancements in cloning, genomics, and numerous molecular biology techniques. Researchers are left with a wide range of powerful downstream applications due to the ability to express recombinant proteins, which they use to further the research studies. The protein expressed on a small scale is majorly used in studying and verifying protein functions. In contrast, those produced on a large scale are significant in studying enzymes, antibodies, and vaccine productions.
To determine optimal cell growth and protein expression conditions for both small and large-scale protein expression systems. Whether a prokaryotic or eukaryotic expression system is required for post-translational modifications, the cell type will significantly impact the tools and reagents needed for optimum protein expression. The following article will enlighten you about five essential things you need to know about protein production.
Protein expression vectors
Expression vectors, also known as plasmids, are circular DNA sequences often utilized by scientists to host the gene encoding their desired protein. Plasmids encoding the desired gene are transformed or transfected into cells to overexpress a protein. Plasmids contain a multiple cloning site (MCS), antibiotic resistance genes for clone selection, unique tags for protein identification and purification, and vital promoter regions to drive protein expression, among other useful elements that facilitate cloning, clone selection, protein expression, and purification.
Protein expression vectors come in various shapes and sizes, with many of these components interchangeable depending on the application’s needs and the cell type utilized for protein expression. Protein vectors aim to ensure they act as promoters’ regions and enhancers, ensuring good transcription of the gene carried in the expression vector. Further, it also provides the efficient production of protein. Protein expression through protein vectors is only achieved by producing considerable RNA, then translated into protein.
Protein expression systems
Bacteria are the workhorse organism for manufacturing recombinant proteins catalyzed by the rapid kinetics and plasmid conversions in E. coli. The 30S and 50S ribosomal subunits of the 70S bacterial ribosome are required for bacterial protein expression. Plasmids harboring antibiotic-resistant genes are employed as a selection tool to detect and isolate bacteria that have adopted plasmids containing the protein-encoding sequence of interest, preventing the growth of plasmid-free cells.
While additional genetic sequencing is frequently required to validate the presence of your gene sequence, plasmid-free bacteria are commonly removed using antibiotics that impede bacterial protein synthesis. The bacterial expression system has a short doubling time, making it convenient for expression host cells. Insect, yeast, and mammalian cell lines are also commonly employed for protein expression. Unlike bacteria, however, eukaryotic cell lines have different molecular machinery for generating post-translational changes (such as glycosylation), often required for protein functionality and useful downstream analysis.
However, it’s good to note that some expression systems, such as bacteria, contain toxic pyrogens, so the synthesized protein should be effectively tested. Therefore, a suitable purification method must be applied during the protein production process.
Purification method
The protein produced with either expression system contains some toxic elements that must be eliminated. The whole protein production process may accumulate unwanted pyrogens that need to be eliminated. Protein purification is the process of removing and filtering protein from a complicated crude mixture. The impurities are always found in the expression host system. To determine the protein structure, function, and interactions, it must first go through the purification process.
The purification process is also essential as it helps divide protein from the non-protein components. The protein separation process from other elements of the produced proteins is usually the tiring and time-consuming part. During the purification process, you can use physic-chemical characteristics of biological activities for the separation process. Protein isolate is the word for the pure outcome. Protein purification is a significant stage in custom protein expression that can be done preoperatively or analytically.
Protein expression conditions
Protein is generated using isopropyl- β-D-thiogalactoside chemical or through the alteration of carbon found in E. coli cells. In both methods, the cells are grown with high densities, especially when exposed to conducive conditions such as baffled shake flask. However, regardless of the cell density, the expressed polymerase should be induced in a mid-to-late log phase, ensuring maximal yield.
This will help avoid any challenges that arise during the stationary such as the generation of proteases. Most expressed proteins will thrive at moderate temperatures and can dissolve at 15–25 °C. These temperature conditions will allow the newly produced proteins to fold properly. Therefore, temperature regulation is significant during induction and the whole production process. If someone wants to know more about protein expression, they can click on the anchor protein expression service.
Recombinant protein expression applications
Recombinant proteins have been changed for maximum protein expression/purification or altered to assess protein function and are encoded within the protein-expressing plasmid. Researchers can use the capacity to add, remove, or change the protein-encoding sequence by a single nucleotide to examine a wide range of fundamental scientific problems and elucidate the protein’s function in both healthy and sick tissues. Recombinant protein expression technology has far-reaching implications beyond basic research and is critical for creating life-saving medicines and vaccines.
Final thoughts
Protein expression is one of the concerns that has attracted researchers’ attention in the biochemistry field. Moreover, it can be defined as how living cells produce and modify proteins. Besides, protein production has also been expressed through laboratory technology. 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.
