The main objective of the proposal is to demonstrate the inactivation of the infective agents implicated in the cause of transmissible spongiform encephalopathies (TSEs) by the action of highly thermostable proteolytic enzymes. This will be measured using the most stringent assay available, namely the intracerebral inoculation of VM mice with protease- treated high litre infectivity stocks from a mouse-passaged strain of the most thermostable TSE variant reported BSE (301 V).The proposal contributes to an EU supported collaboration involving, the group of Prof Garo Antranikian in Hamburg, who have considerable expertise in the purification and characterisation of thermostable proteases from hyperthermophiles and the group of Prof. Mose Rossi in Naples who have expertise in the cloning and expression of genes from hyperthermophiles to permit large scale production.
The proposal involves the growth of a diverse range of thermophilic, thermoacidophilic, hyperthermophilic and thermoalkalophilic microorganisms from both the archaeal and bacterial domains. CAMR have already developed novel strategies for the cultivation of thermophiles and hyperthermophiles to high cell densities which will allow production of sufficient material for the initial characterisation of extracellular proteases. This will enable the effect of high temperature proteolysis on the inactivation of TSE agents to be studied over a wide range of pH. A breeding colony of mice will be established which will be used to provide a supply of high titre infectious mouse brain for inactivation tests.
Purified proteases will be screened for pH and temperature optima, sensitivity to protease inhibitors and substrate specificity. Optimised conditions will then be used to study the proteolysis of high-titre mouse passaged BSE agent present in the infected mouse brain homogenate. This will be monitored by 2D-gel electrophoresis of total protein looking for elimination of the spot associated with the misfolded form of PrP. Mouse brain homogenates showing significant depletion of the abnormal protein will be tested for complete inactivation of the TSE agent by intracerebral injection into mice. Those enzymes indicating inactivation of TSE agents will be cloned by the group of Mose Rossi to enable larger quantities of enzyme production for use in subsequent studies using infected bovine brains.
Currently, as recognised by MAFF, inactivation of TSE agents is an intractable problem, whether in medical or veterinary research, food handling and processing, or the pharmaceutical and cosmetic industries. Reliable inactivation of TSE agents using highly thermostable proteases will be a major advancement in several areas. For processes involving potentially infected animal materials (food, pharmaceutical and cosmetics industries), it will allow the safe recovery of non-proteinaceous components rather than their destruction. It will also permit the introduction of effective non-destructive disinfection cycles for most standard equipment used in handling and processing. In medical and veterinary applications, highly thermostable proteases will provide a research tool for the characterization of causative agents of TSEs and allow a greater range of techniques to be used in their study, since dedicated equipment will not need to be destroyed after use.