Building Better Recombinant Antibody Factories
For decades, scientists have used the tried and true workhorse of molecular biology, Escherichia coli, for recombinant protein expression. While these bacteria are supremely adaptable, inexpensive to culture, and capable of forming miniature factories for the synthesis of various molecules, there are some problems associated with their use.
Molecular biology research often involves the purification of recombinant molecules produced in bacteria (e.g. plasmids, proteins, etc.) to test their in vitro effects on target cells, often mammalian cells lines. However, harvesting molecules from bacteria has some drawbacks, preeminent among them are endotoxins. Endotoxins (lipopolysaccharide, LPS) are molecules found in the outer membrane of Gram-Negative bacteria that activate innate immune cells, induce cellular stress, and cause pyrogenic effects in mammals. If endotoxins are not eliminated they can complicate cell culture-based experiments and in vivo studies by introducing experimental variations unrelated to the effects of the molecules prepared from bacteria (e.g. plasmids, proteins, etc.).
Another major deficiency is that bacterial cells lack many of the natural protein modification systems found in eukaryotes. Therefore, human gene-derived recombinant proteins expressed in bacteria will produce proteins that may lack post-translational modifications required for proper function.
With the development of high yield transient mammalian expression systems, producing recombinant proteins in mammalian cells has become a cost-effective option for academic labs. Expressing recombinant proteins in human cell culture, instead of E. coli, offers several major advantages. First, human cells can produce properly-modified antibodies, rather than bacterially-derived, unmodified variants. Second, endotoxins are not collected when harvesting the antibodies. Finally, the cultured cells can produce larger recombinant antibodies efficiently. Together, these advantages are prompting researchers to reconsider the cell type in which they produce recombinant mammalian proteins.
A recent paper 1 published in Nature Protocols presents an efficient method for recombinant human antibody production using cell culture. To achieve this, Vazquez-Lombardi and colleagues used the ZymoPURE plasmid purification system to prepare endotoxin-free recombinant antibody mammalian expression vectors which were then transfected into HEK293 cells using a cationic lipid-based transfection reagent. Following transient expression, the recombinant antibodies were harvested and characterized. This method greatly simplifies the production of high-quality recombinant antibodies for use in mammalian systems, making it ideal for generating multiple recombinant antibodies in an academic setting.
Reevaluating and improving core procedures, such as antibody production, is an exciting and growing area of research that holds much promise for improving basic molecular and biochemical techniques. As demand for cell culture-based protein production increases, the future will rely on sophisticated methodology that incorporates transfection-ready plasmid purification.
Read the article here.
1. Vazquez-Lombardi, R., Nevoltris, D., Luthra, A., Schofield, P., Zimmermann, C., Christ, D. 2018. Transient expression of human antibodies in mammalian cells. Nature Protocols 13(1): 99-117.