Production of Recombinant Proteins

For recombinant protein production in E. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the product. The present paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth rate has been analyzed, in order to understand and define possible modifications with influence on post-induction cellular behavior. A constant addition profile has been demonstrated to render higher specific aldolase production than the exponential addition profile, probably due to a more constant environment for the cells. On the other hand, amino acid (leucine) supplementation has proven to increase protein quality in terms of activity units (U) per unit mass of RhuA (U mg(-1) RhuA), alleviating metabolic overload. Based on the above, a production process was set up and scaled up to pilot plant. Resulting production was double that of a standard laboratory operation, 45,000 U L(-1), and almost all the protein retained the 6xHis-tag with the highest quality, 11.3 U mg(-1) RhuA.

Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more "human-like" post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

Stem cell research is well known in clinics to treat diseases as huge research has resulted in successful isolation, maintenance and manipulation of stem cells in vitro. Much information regarding molecular biology of number of cells/stem cells is known and have shown a promising potential in biotechnology. Biotechnologist have used novel approaches in last decade for the production of human compatible recombinant proteins and recently, stem cells have been considered as potential biotechnological agents. Focus should be done to engineer cells for specific trait production such as germ cells. It also has been shown that maintenance of germ cells is not an easy task but successful trans-differentiation of other multipotent stem cells such as mesenchymal stem cells toward germ cell linages have opened the opportunities to engineer MSC and then trans-differentiate towards germ like-cells which can be used to develop transgenic animals. Focus should be done on cell based biotechnological productions to revolutionize health biotechnology.