Cytoplasmic Protein Folding Technology

“Patented” Cytoplasmic Oxidative Protein Folding

Cytoplasmic protein folding is a propriety technology under license to Paras Biopharmaceuticals, for the high level expression of ho­mogeneously folded disulfide bond containing proteins in the cytoplasm of E. Coli. The technology offers all of the cost benefits of expression in E. Coli rather than in eukaryotic systems, while at the same time retaining the high yields obtained in cytoplasmic expression. The biologics produced are of the highest quality and have maximal biological activ­ity, unlike the activity levels obtained for many secreted eu­karyotic proteins when expressed in the cytoplasm without CyDisCo technology.

Native disulfide formation is a complex multi-factorial proc­ess and has not been possible in the cytoplasm of E. Coli due to the presence of two reducing pathways. The synthesis of disulfide bond containing proteins is therefore either done in eukaryotic cells, in the periplasm of bacteria or by using in vitro oxidative refolding methods. All of these have prob­lems relating to cost and efficiency.

Advantages of Technology

Technology allows:

Protein Folding Technologies

Disulfide bond formation is a post-translational modification that is required for the correct folding and function of around one-third of human proteins, including most secreted and outer-membrane proteins. Proteins that include disulfide bonds include: insulin, antibody fragments, interleukins, interferons, erythropoietin and many others.

Native disulfide formation is a complex multi-factorial proc­ess and has not been possible in the cytoplasm of E. Coli due to the presence of two reducing pathways. The synthesis of disulfide bond containing proteins is therefore either done in eukaryotic cells, in the periplasm of bacteria or by using in vitro oxidative refolding methods. All of these have prob­lems relating to cost and efficiency.

New Technology Developments

Unlike previous systems, this works in any E. Coli strain and does not require the disruption of the reducing pathways naturally present. The system also works in all media and does not require any supplementation of the media, reducing costs.

The system has been successful for the production and pu­rification of more than 50 eukaryotic proteins, in yields up to 100mg/L from shake flasks and in excess of 1g/L in fer­mentation using unsupplemented minimal media.

High Level Protein Production

Production of human growth hormone 1 in yields of more than 1g/L of purified protein from standard fermentation. Paras Biopharmaceuticals is currently combining this CyDisCo technology with our own BioMultiFold® to further increase yields.

The proteins produced are homogeneously folded, have native disulfide bonds and full biological activity. By avoiding time-consuming and inefficient in vitro refolding, the use of CyDisCo significantly reduces production costs and development time.

Expertise at Paras Biopharmaceuticals

Paras Biopharmaceuticals Finland Oy has an unrestricted license to use CyDisCo for the development of its own products and in the development of production processes for other companies. Furthermore, Paras Biopharmaceuticals Finland Oy has in-house expertise in CyDisCo technology to ensure that the most appropriate variant is used to further cut the cost and time of development.