Protein Expression
Advancements in genomics, cloning, and a wide array of molecular biology techniques have enabled researchers to express heterologous proteins in various biological systems. The ability to express recombinant proteins has opened up numerous powerful downstream applications, providing researchers with valuable tools for further research studies. At small scales, overexpression of proteins can facilitate studies focused on understanding protein function, while large-scale protein production is essential for the manufacturing of enzymes, antibodies, and vaccines. Identifying optimal conditions for cellular growth and protein expression is critical for both small- and large-scale protein expression systems. Whether a prokaryotic or eukaryotic expression system is required for post-translational modifications, the choice of cell type will largely determine the tools and reagents needed to achieve optimal protein expression,
Overview
Protein Expression Vectors
Expression vectors or plasmids are circular DNA sequences commonly used to host the gene encoding the protein of interest. Once the plasmid containing the gene is transformed or transfected into cells, it facilitates the overexpression of the protein. These plasmids are designed with several essential features, such as a multiple cloning site (MCS), antibiotic resistance genes for clone selection, unique tags for protein identification and purification, and strong promoter regions to drive protein expression. There are various protein expression vectors available, as many of these elements are interchangeable to suit specific application needs and cell types used for protein expression.
Bacterial, Mammalian, and Additional Protein Expression Systems
Bacteria, particularly E. coli, are commonly used for recombinant protein production due to their fast growth and easy plasmid transformation. In bacterial systems, protein expression is driven by the bacterial ribosomal subunits (30S and 50S) of the 70S ribosome. Plasmids carrying antibiotic-resistant genes are used to select bacteria that have successfully incorporated the gene of interest. Other expression systems, such as insect, yeast, and mammalian cell lines, are also used depending on the requirements of the protein, particularly when post-translational modifications, such as glycosylation, are needed for protein functionality.
Recombinant Protein Expression Applications
Recombinant proteins are encoded by protein-expressing plasmids, which can be modified for optimal protein expression and purification or mutated to study protein function. The ability to alter protein-encoding sequences allows researchers to investigate fundamental biological questions and explore protein function in both healthy and disease tissues. Recombinant protein expression technology is essential not only for basic research but also for the development of life-saving therapeutics and vaccines.