Advances in Improved Expression of Recombinant Proteins in Microbial Systems
Among the biomolecules in use or in testing are nucleic acids (i.e., plasmid or viral DNA and RNAi for gene therapy) and polyssaccharides (i.e., antigens), although the bulk of products are proteins and peptides. Synthetic protein production by in vitro processes is an expanding field, with significant yield improvements compared to historically low yield procedures (Kim and Swartz, 2003), but is likely to be very expensive at scale. Due to biology, history and economics, almost all production utilizes the ability of cultured cells to produce natural or recombinant proteins. Numerous organisms have been evaluated, from prokaryotes (bacteria, etc) to viruses to insect to eukaryotes (i.e., yeast, mammalian cells, whole animals) to plants. Some products are natural and harvested from cultivated cells (i.e., mAbs, other natural), or from an animal (Abs from livestock), but most products are produced by engineering an expression system and gene to a host cell to manufacture a desired protein. Advances in our understanding of the cellular mechanisms coupled with enhancements in technologies have resulted in a significant decrease in the cost of goods associated with the production of biomolecules. With newer and more advanced technological discoveries, such costs may continue to drop, as the bio-therapeutic market continues to grow. In fact, it is anticipated that the bio-therapeutic market will double within the next 10 years. Recent advances have resulted in whole animal biofactories and other technologies. The primary factory for the production of bioproducts, however, is still the cultured single cell. E. coli has historically been one of the primary expression systems for recombinant proteins. In choosing an expression system, a user will select a host organism, a promoter and often will choose which, if any, tags will be employed, often from the numerous vectors available from molecular biology companies (large and small) and academic research institutions. There are important implications of selection marker related to human use products. Another major factor in the choice of expression systems are the patents held for many of these expression systems and the legal ramifications of their use should a product leave the research phase and enter the commercial phase. There are a wide variety of tags and fusion peptides available with E. coli expression systems. Many of these are designed to provide a convenient, quick and inexpensive method of product purification at the bench scale. An alternative approach to the use of plasmid vectors is to integrate the gene, with a suitable promoter, directly into the bacterial genome, typically including a selection marker. Yeast systems have been a staple of production of large amounts of proteins for industrial and biopharmaceutical uses for many years. Recombinant proteins in yeast can be over-expressed so the product is secreted from the cell and available for recovery in the fermentation broth. Proteins secreted by yeasts are heavily glycosylated at consensus glycosylation sites. The ability to produce large quantities of humanized glycoproteins in yeast could offer advantages in that glycosylated structures could be highly uniform and thus easily purified. A few other fungal systems have also been successfully used for recombinant protein production. Some of these systems are based on non-fermentation yeast species like Pichia methanolica and Hansenula polymorpha.
Note: This chapter was originally published in the first edition of this study, in its entirety.
About the Authors
Daniel Rudolph, Ph.D.
Dr. Rudolph joined Cambrex Bio Science Baltimore in 2000 as a Process Engineer in the Process Development Group. Since joining Cambrex, Dr. Rudolph has been involved in projects ranging from tech transfer, strain development, upstream process development and scale-up, to cGMP production. Prior to joining Cambrex, Dr. Rudolph was a Research Scientist with SymBiotech, focusing on assay development, engineering bacteria, and yeast strains for the study of gene expression in addition to development and construction of automated medical devices. Dr. Rudolph holds a B.S. and M.S. degree in Chemical Engineering from Bucknell University and a Ph.D. in Chemical Engineering from the University of Virginia.