Projects / Programmes
The development of enzyme formulation for disinfecting prion-contaminated areas.
| Code |
Science |
Field |
Subfield |
| 4.06.06 |
Biotechnical sciences |
Biotechnology |
Finishing processes in biotechnology |
| Code |
Science |
Field |
| B510 |
Biomedical sciences |
Infections |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
prions disinfection, thermostable proteases, penisin, heterologous expression system, Streptomyces sp.
Researchers (18)
Organisations (3)
Abstract
Transmissible spongiform encephalopathy (TSE) forms a family of neurodegenerative diseases known for animal and humans. The emergence of a variant form of human Creutzfeldt-Jakob disease (vCJD), as a result of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, has increased the profile of transmissible spongiform encephalopathies, as they have become a serious risk to public health policy worldwide. CJD is a fatal neurodegenerative disease that affects on average 1 person per million per year in the United States. Infective prions that cause CJD or other TSEs show high resistance to conventional chemical and physical decontamination methods. Prions are well-known to bind tightly to surfaces, including stainless still surfaces.
The basic procedures for preventing iCJD transmission via surgical instruments are sterilisation or prion decontamination according to guidelines by autoclaving them at high temperatures (from 121°C to 132°C and using sodium hydroxide. These procedures usually take around 1 hour, but surgical equipment (eg. endoscopes) cannot resist these harsh conditions. Efficient removal of biofilms from medical devices is another big challenge in healthcare, to avoid hospital-acquired infections. Again, there can be problems with delicate devices like flexible endoscopes, which cannot be cleaned using harsh chemicals or high temperatures.
There are a few alkaline and enzymatic detergents that can reduce the prion infectivity. Although that there are rare proved transmissions of CJD, as they so called iatrogenic CJD (iCJD), through medical equipment has been rare, the consequences can be far-reaching. With the evidence of tens of thousands infectious carriers just in the United Kingdom, there is increasing concern of potential transmission of infection. The long incubation periods for CJD, along with the need for diagnosis of CJD through brain tissue, might indeed are causes that can possibly lead to uncontrolled CJD transmission via contaminated surgical instruments.
In 2011, we patented a procedure for enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human), as well as aggregates related to Alzheimer’s disease. However, very low productions yield of pernisine, at ( 0.5 mg per 1 L of culture, has hampered its industrial applicability. Our breakthrough with an Escherichia coli expression system through codon optimization of the gene coding for pernisine can now provide ) 20-fold increases in yields, which has opened new opportunities for industrial purposes. However, the need for the activation of recombinant pernisine and the secretion of this protein extracellularly has raised new obstacles for industrial applications. Our proposed solution is to use a relatively new expression system with Streptomyces sp. and to make use of a shorter version of the pernisine gene, which is constructed without its proregion.
The aim of the project is to develop this new and efficient formulation for cleaning/disinfection the prion's contaminants through a system based on recombinant pernisine, which will be carried out in collaboration with Borer Chemie AG, as one of a leading supplier of products for aqueous cleaning and disinfections.
Significance for science
Original scientific contribution: The applied research approach adopted within this project is itself novel and will significantly extend our understanding of the mechanism of action and physicochemical and biological properties of the archaeal protein pernisine expressed in Streptomyces. This will allow the development of a new formulation to disinfect and clean the prion contaminants in laboratories, and for surgical instruments (Poklar N. Vilfan T. 2012). The project is design so it contains the application note based on science. The scientific results obtained will be published in peer-reviewed journals. The impact of the original scientific project’s will be in-vitro validation of new formulation of pernisine at Borer Chemie AG. As well as development of the new formulations, the products also will also be improved in terms of their compositions. All of these properties are essential for high added value products.
The main scientific contributions:
(i) Generating the basic knowledge of the mechanism of PrPSc and other TSE degradation processes by the thermostable protease pernisine
(ii) Knowledge of using the Streptomyces sp. as a heterologous expression system for archaeal proteins
(iii) Understanding the mechanism/correlation between thermal stability and activity of proteins found in hyperthermophilic archaea.
(iv) 3D structure of recombinant thermostable serine protease (pernisine) should enable the further optimisation of the enzyme activity
(v) Development of different enzyme-based formulation(s) for the decontamination of PrPSc or other protein aggregates
Significance for the country
With the presented project, we would like to use pernisine, which has shown potential for industrial applications as a prion decontaminating agent with a new expression system of Streptomyces sp. to use in a newly developed formulation. The results will be tested, along with the development and commercialisation, and these will have a rapid impact on the economic benefits for the collaborating partners. Potential enzyme-based new formula(e) can be used for cleaning of potential PrPSc infections in medical fields (hospitals, medical centre), to avoiding the fatal consequences of TSE. Our two already granted patents (one is European: Compositions and Methods for Degradation of Protein Deposits and Prions; another is Slovenian: Overproducing recombinant pernisine in heterologous expression systems) will have beneficial financial impacts. The development of technology for Streptomyces sp. as an expression system for producing pernisine can be further upgraded to a new patent. Successful collaboration can further result in a newly establish company for developing the new industrially important proteins or producing pernisine. Some preliminary results are already promising for new articles with relatively high impact factors. The biotechnology master students and PhD students will be involved in the research work. The partnership involved in the project will be strengthen on the basis of future collaboration and knowledge transfer between industrial and academic partners. Successful collaboration with industry can lead to further collaboration on similar application projects with other different promising enzymes from A. pernix.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
