Patent Grant
11407988
This application is the 35 U.S.C. § 371 national stage application of PCT Application No. PCT/EP2016/082557, filed Dec. 23, 2016, where the PCT claims priority to GB. Patent Application Serial No. 1522814.1, titled “COMPOSITION AND USES THEREOF” filed on Dec. 23, 2015, both of which are herein incorporated by reference in their entireties.
The Sequence Listing submitted Feb. 12, 2022, as a text file named “2022-02-12_Substitute_Sequence_Listing_AYL-00001-U-US-01_ST25.txt” created on Feb. 11, 2022, and having a size of 27,750 bytes is hereby incorporated by reference.
The invention relates to novel proteases for degrading proteinaceous infectious agents (prions).
Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders with the ability to affect both animals and humans and include scrapie, Bovine Spongiform Encephalopathy (BSE) and chronic wasting disease (CWD) in animals and Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome and Fatal Familial Insomnia in humans. These disorders, which are generally associated with the accumulation in the brain of an abnormal partially protease resistant isoform (PrPSc) of the normal endogenous prion protein (PrPC), can be infectious, genetic or sporadic in origin.
There is continuing concern over the potential to transmit prion diseases in the hospital sector through the use of contaminated instruments. These disorders are generally associated with the accumulation in the brain of an abnormal partially protease resistant isoform (PrPSc) of the endogenous prion protein (PrPC). The existence of asymptomatic TSE carriers has led to concerns over the risk of iatrogenic TSE transmission, which has already occurred from variant Creutzfeldt-Jakob disease (vCJD) carriers. From a re-analysis of appendices samples resected from patients prior to their vCJD development, it was identified that vCJD could be detected in patient samples at least 2 years before the development of vCJD symptoms. What complicates the aspect of asymptomatic carriage is that the length of time it takes for the development of vCJD symptoms appears to be dictated by the PRNP codon 129 polymorphism (methionine (M)/valine (V)). This also makes it difficult to predict the potential level of vCJD. Recently, it has been found, through the immunohistochemical analysis of stored paraffin-embedded appendix samples identified an overall prevalence of abnormal prion in 493 per million of the population (or approximately 1/2000 are carriers). In addition, considering that so far all definite and probable cases of clinical vCJD have been homozygous for methionine at position 129, bar one possible vCJD case who was heterozygous (PRNP 129 MV), it is a concern that studies identified a higher than expected prevalence of abnormal prion cases in appendix samples of those that were homozygous for valine.
The potential implications of the asymptomatic carrier for secondary vCJD transmission drives the need for appropriate measures to prevent such transmission. Even when patients present with early vCJD symptoms, the signs of this disorder can vary significantly between patients. Compounding the difficulties for clinicians is that what appears to be non-CJD related morbidities results in misdiagnosis, in such instances diagnostic tests to aid in diagnosis would help with identification of suspected cases and minimise transmission possibilities. However, such diagnostic tests may be limited in the asymptomatic stages of disease and appropriate mild decontamination techniques can be significant to the control of spread in the hospital sector.
Ultimately, due to the delicate nature of surgical equipment the ideal prion decontaminant should work under mild conditions. However, PrPSc is resistant to standard sterilisation guidelines (such as those issued by the World Health Organisation (WHO) or The Society for Healthcare Epidemiology of America (SHEA)), and practises that are advised to sterilise equipment are simply too severe. For example, some methods proposed to eliminate prion contamination, including exposure to NaOH (1N), sodium hypochlorite solution (20,000 ppm of available chlorine) or high temperature porous load autoclave, are not suitable for many delicate medical devices. A three stage destruction involving heating in 4% SDS at 100° C. followed by proteinase K digestion and then pronase digestion was proposed as a method to lower the risk of prion transmission. This process lowered vCJD signal in infected brain to an undetectable level, and it almost completely eliminated Rocky Mountain Laboratory (RML) scrapie infectivity from contaminated steel wires that were subsequently inoculated into Tg20 transgenic mice and wild-type CD-1 animals. Modified mixes such as alkaline cleaners (pH 12.2) along with mixes of 0.2% SDS and 0.3% NaOH (pH 12.8) have also been found to completely removed scrapie strain 263 infectivity. For delicate equipment, however, there is a need for more harmless approaches lacking high temperature and/or alkaline processing and the processes advised by the World Health Organisation for prion decontamination are too severe for most reusable hospital equipment. The effect of these treatments on some medical equipment have been studied and evident damage was seen on the items tested. Furthermore, a study of ready to use reusable surgical instruments identified that the level of residual protein remaining on these devices could pose a risk for the transmission of the prion agent. It has also been reported that none of the commonly used enzymatic cleaners employed in Sterile Service departments allowed for complete removal of protein or prion matter from test material. Eliminating protein load on instruments is essential not only for reducing the risk of prion transmission but also for controlling other hospital-acquired infections. There is therefore a problem with existing approaches and that is the lack of suitable processes for delicate equipment.
WO2008057293 discloses compositions and methods for prion degradation, decontamination or disinfection. The composition described comprises an oxidizing agent, one or more proteases and a surfactant such as an ionic surfactant/detergent. However, the composition requires a number of different components which would have different storage and handling requirements and/or applied in a time consuming sequential manner.
Relatively few microorganisms have been previously reported to produce a protease with the ability to degrade PrPSc. Müller-Hellwig et al., (2006) Syst Appl Microbiol 29: 165-171 describes screening over 600 bacterial isolates for protease production, of these 199 secreted a protease and only 6 were found to have some level of prion degrading activity against scrapie infected hamster brain homogenate. Hui et al., (Hui et al., (2004) In Vitro Cell Dev Biol Anim 40: 293-296) screened over 500 microorganisms to obtain the Streptomyces sp that secreted the protease E77, it degraded scrapie infected hamster brain homogenate at 60° C. and pH 11.0. Greater than 300 bacterial strains were screened to obtain the Bacillus sp. MSK103 protease (Yoshioka et al., (2007) J Appl Microbiol 102: 509-515). The ability of a protease to breakdown prion material is thought to be dependent on conditions that open up the protein structure and enable the protease to access the protein backbone. As a consequence a number of proteases have been tested for their anti-prion action at alkaline pH. The alkaline pH is also chosen as the pH itself contributes to inactivation of the prion agent.
There is also a problem with existing approaches in dealing with environmental contamination. For example in the agricultural industry, the prion agent has been detected in urine (Sc237 strain) and blood of infected animals (263K strain), and in the faeces of symptomatic and asymptomatic deer which risks the continued transmission of animal TSEs. Both scrapie of sheep and CWD of elk and deer are horizontally transmissible, and their agent can remain infectious for years in the environment.
The persistence of the prion agent within the environment and the demonstrated transmission of CJD through surgery and contaminated products emphasises the need for decontamination approaches that can be used in a safe and routine manner to limit the spread of TSEs.
It is therefore an object of the present invention to mitigate or address one or more of the above identified issues with previous prion degradation techniques. It is a further object to identify a composition which can be used for prion degradation, decontamination or disinfection. Ideally, such a composition would be functionally active at less severe conditions than previously described so that it could be used in conjunction with medical equipment and especially more delicate medical equipment. Additionally, or alternatively, the composition may be suitable for use in environmental and/or agricultural prion degradation, decontamination or disinfection. It would be desirable if the composition was relatively easy to produce, handle and use for prion degradation, decontamination or disinfection.
In accordance with a first aspect of the present invention, there is provided a composition for degrading prion material comprising a Thermitase.
The term “degrading” is intended to mean to breakdown or reduce the activity or infectivity of a prion or prion material which would otherwise have the potential to result in the neurodegeneration (or a quicker onset of neurodegeneration) in an exposed animal (including humans). The term also includes disinfection and decontamination of a substrate from prion or prion material.
Advantageously, it has been found that Thermitases can degrade prion material without additionally requiring other components such as oxidising agents or surfactants.
It is preferred that the Thermitase has the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4, or a sequence or sequences having 95% or more homology thereof. More preferably, the Thermitase has the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4, or a sequence or sequences having 97% or more homology thereof. Even more preferably, the Thermitase has the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4, or a sequence or sequences having 98% or more homology thereof. Most preferably, the Thermitase has the amino acid sequence of SEQ ID NO: and/or SEQ ID NO: 4, or a sequence or sequences having 99% or more homology thereof. Ideally, the Thermitase has the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 4.
Homology may be determined using on-line homology algorithm “BLAST” program, which is currently publicly available at http://www.ncbi.nlm.nih.gov/BLAST/. Sequences which have a high degree of homology may be mutants, variants or genetically modified sequences. Deliberate alteration of amino acid sequences may be accomplished by conventional (in vitro) genetic manipulation technologies, such as gene disruption, conjugative transfer, etc. Genetic modification includes introduction of exogenous and/or endogenous DNA sequences. Natural or induced mutations may include at least single base alterations such as deletion, insertion, tansversion or other DNA modifications which may result in alteration of the amino acid sequence encoded by the DNA sequence.
The composition may further comprise a number of other constituents which are often required to aid storage and/or ensure the appropriate activity of an enzyme. Preferably, the composition further comprises a buffer. Such a buffer may be a biological buffer such as HEPES.
The composition will preferably have a pH in the range of about 6 to about 13. More preferably, the composition will have a pH in the range of about 7.5 to about 12.5. Most preferably, the composition will have a pH in the range of about 7 to about 12.
The Thermitases will preferably have a wide activity temperature range. The Thermitase will preferably be active in the range of about 10° C. to about 65° C. More preferably, the composition will be active in the range of about 15° C. to about 60° C. More preferably, the composition will be active in the range of about 15° C. to about 55° C.
The composition may be formulated in the form of a solution or is formulated so as to easy form a solution if desired. The skilled addressee will understand how various enzyme solutions can be prepared for immediate use or for subsequent dilution or rehydration. Preferably, the solution is an aqueous solution. Most preferably, the composition comprises the Thermisase component in a lyophilised activated state.
In some instances, it may be preferred that the composition comprises two or more Thermitases. This potentially increases the activity range of the composition at successfully degrading prion material. Preferably, the composition comprises Thermitases having an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 4, or sequences having 95% or more homology thereof.
The composition of the present invention will have wide application wherever prion material requires degradation. For example, the composition may be for use in degrading prion material on medical equipment. The composition is particularly suited to delicate medical equipment which is often damaged by the use of high temperatures and/or highly alkaline sterilising products. Alternatively the composition may be for use in degrading prion material in the environment, such as agricultural land.
The composition may be in the form of a ready-made solution or spray. Alternatively, the composition may be in the form of a pre-mix solution which is for subsequently diluted or added to a liquid (such as an existing medical equipment sterilising solution) for application to degrade prion material.
In accordance with another aspect of the present invention there is provided a protease having the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 or an amino acid sequence having 98% or more homology thereof.
More preferably, the protease has the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 or an amino acid sequence having 99% or more homology thereof. Most preferably the protease has the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.
The protease may be for use in degrading prions or prion material.
In accordance with yet another related invention, there is provided a method for prion degradation, decontamination or disinfection, the method comprising contacting a substrate containing and/or coated with prion material with a composition comprising one or more Thermitases.
It is preferred that the method is conducted under conditions effective to enable the activation or activity of the Thermitase to degrade prion material. Preferably, the method is conducted in the range of about 10° C. to about 65° C. and/or a pH in the range of about 6 to about 13. More preferably, the method is conducted in the range of about 15° C. to about 60° C. and/or a pH in the range of about 6.5 to about 12.5. Most preferably, the method is conducted in the range of about 10° C. to about 65° C. and/or a pH in the range of about 7 to about 12.
The Thermitase will preferably comprise the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 or an amino acid sequence having 98% or more homology thereof. More preferably, the Thermitase comprises the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 or an amino acid sequence having 99% or more homology thereof. Most preferably, the Thermitase comprises the amino acid sequence of SEQ ID NO: 1, SED ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.
In another yet related aspect of the present invention, there is a method of producing a Thermitase for use in prion degradation, the method comprising transforming a host organism with a DNA sequence comprising SEQ ID NO: 3 or SEQ ID NO: 6 or a DNA sequence having 95% or more homology thereof, and putting the DNA sequence in the host under a positive promoter control for stimulating production of a Thermitase in the host organism and harvesting said Thermitase.
The steps of transforming a host organism with DNA so as to produce the desired enzyme under positive promoter control will be commonly used recombinant genetic manipulation techniques which will be well understood by the skilled artisan.
In accordance with a further aspect of the present invention, there is provide a kit of parts for prion degradation, decontamination or disinfection, comprising:
Such a kit may further comprises a container housing the Thermitase and/or buffer solution or buffer solution constituents, a means for measuring and/or mixing the correct quantities of Thermitase and/buffer solution or buffer solution constituents together so as to form a solution which can degrade prion material.
Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures, in which:
A number of historical University College Dublin microbial isolates were screened for enzyme systems with the ability to degrade pK resistant PrP (PrPSc) under mild conditions. Initially Bacillus and Actinomycete spp were screened for protease activity, of those that were screened both the thermophilic actinomycete 16 and 10 were found to have protease producing abilities.
Screening Programme to Identify Protease Producing Bacterium
The Thermoactinomycete sp. 16 was selected after a screening programme of a range of Bacillus sp and Actinomycetes. Table 1 below shows the results for investigating protease producing Bacillus isolates.
Bacillus isolate
The isolates were grown at 200 rpm at 55° C. up to 48 h. The CFS was assayed for protease activity using BSA (1% w/v) in 0.1M phosphate buffer pH 7.0.
Table 2 below details the screening and selection of protease producing Actinomycete isolates.
Streptomyces
aminophilus
Thermoactinomyces
Thermoactinomyces
As can be seen from Table 2, of the micro-organisms screened, protease production was only seen in two Thermoactinomyces sp. No 10 and 16. Both of these species were isolated. Although the actinomycetes are known for their enzyme production, not all produce proteases. Thermoactinomyces sp. 16 was chosen initially for further work over Thermoactinomyces sp. 10 as its enzyme levels in the cell free supernatant (CFS) were maintained between 24 and 48 h in the screening studies, potentially indicating better enzyme stability. When conditions of enzyme production from Thermoactinomyces sp. 16 at 40° C. were optimised, maximal enzyme levels were produced at 72 h growth and these levels were maintained for at least up to 168 hours (as shown in
The first potential candidate protease was identified from a Thermoactinomyces isolate sp 16 and was denoted protease 16. Protease 16 was later characterised as a Thermitase. This protease was found to have the ability to degrade pK resistant prion material under mild conditions and allowed for complete loss of detectable prion signal from 50 μg protein, and it did so within 15 min at 22° C.
The Thermoactinomycete isolates 10 and 16 were isolated from mushroom compost using International Streptomyces Project medium no. 4 (ISP4). Thermoactinomyces sp. 16 and 10 are Gram-positive bacterium. The organisms grew on a nutrient agar ((g/l) soluble starch, 10.0; Lab-lemco agar, 23.0; yeast extract, 2.0, pH7.2)) forming colonies that were initially firm compact and leathery. The colonies were then completely covered with aerial mycelium, which was velvety in texture and colonies displayed sectoring similar to that of Streptomyces griseus. The reverse side of colonies on the nutrient agar and ISP4 were brown/yellow and brown, respectively. Light microscopy revealed the aerial mycelium consisted of straight to flexuous spore chains consisting of greater than 12 arthrospores. The actinomycete isolate was identified as belonging to the Thermoactinomycetes by virtue of the isolates macroscopic and microscopic characteristics. The thermophilic nature identifies them as Thermoactinomycetes.
A deposit of the Thermoactinomyces sp 16 strain was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on Aug. 18, 2010 and accorded the accession number NCIMB 41754.
The protease of a second Thermoactinomyces sp isolate sp 10, which produced a protease denoted 10 was found to also degrades the prion within 15 min at 22° C.
Protease Production
The microbial isolate 10 and 16 were maintained at 4° C. on slopes of nutrient agar containing 1% (w/v) starch at pH 7.0. To produce the protease, a standard inoculum was developed by transferring a loopful of culture to 50 ml inoculum medium (g/l): soluble starch 30.0, bacto-peptone 5.0, yeast extract 5.0, CaCl22H2O, 0.5, MnCl24H2O 0.5, MgCl26H2O 0.48, KH2PO4 1.0, pHi 7.0. The culture was grown for 48 h at 55° C. and 200 rpm. 5% (v/v) standard inoculum was transferred to 50 ml production medium (g/l): maltose 30.0, bacto-tryptone 5.0, yeast extract 5.0, CaCl22H2O 0.5, MnCl24H2O 0.5, MgCl2 6H2O 0.48, KH2PO4 1.0, pHi 7.0. The cells were then grown at 40° C. and 200 rpm for 72 h. Cells were then removed from the culture by centrifugation at 10,000 g for 15 min. The protease activity in the cell-free supernatant was then determined.
Purification of the Protease 16
The secreted protease was purified from cell free supernatant by (NH4)2SO4 (0-60%) fractionation followed by affinity chromatography on Bacitricin-Sepharose 4B according to the quantities shown in Table 3 below.
The first step was ammonium sulphate (0-60%) fractionation. To express the final ammonium sulphate concentration as % ammonium sulphate saturation the Green and Hughes calculations were used. The precipitate obtained after fractionation was suspended in 0.02M HEPES buffer, pH 7.0 and was dialysed against water for 24 h. The second step used was affinity chromatography on bacitracin-Sepharose 4B using a modification of the method of Stepanov and Rudenskaya. Bacitracin-Sepharose 4B was equilibrated with 0.04M Tris/HCL, pH 7.9. Enzyme solution in 0.04M Tris/HCL pH 7.9 was applied to the column and the column was washed with the same buffer to remove all unbound protein. The protease was then eluted from the column with 25% (v/v) propan-2-ol in 0.04M Tris/HCL pH 7.9, containing 1M NaCl at a flow rate of 0.5 ml/min. The propan-2-ol and NaCl were removed from the protease solution by dialysis.
Protease Assay
In the protease assay, activity was determined by the addition of 50 μl enzyme to 50 μl bovine serum albumin (BSA) (1%, w/v) in 0.1M CAPS buffer, pH 11.0 and incubated for 30 min at 40° C. The reaction was then stopped by the addition of 200 μl 5% (w/v) trichloroacetic acid (TCA). After 10 min the precipitate was centrifuged at 3,000 g for 10 min. Total protein concentration in the supernatant was measured using the bicinchoninic acid (BCA) protein assay kit (Sigma). A unit of activity was defined as the amount of enzyme releasing 1 mg TCA soluble protein fragments from BSA in 30 min at 40° C.
Protease Characterisation
For determination of the effect of pH on enzyme activity the protease assay was carried out using BSA (1%, w/v) prepared in 0.1M Briton and Robinson's Universal buffer; pHs 2.0-12.0 and the assay was performed as above. As shown in Table 3, recovery of approximately 50% after purification was achieved. From a pilot scale shaker flask production from one run resulted in a yield of 3000 units, after purification this would reduce to 1500 units. The purified protease was an alkaline protease with optimal activity towards pH 11.0 and it displayed highest resistance to pHs between pH 8.0 and 11.0. The enzyme was optimally active at 50° C. and exhibited high stability up to 50° C. after 1 h exposure (as shown in
Both pefabloc and phenylmethylsulfonyl fluoride significantly inhibited the enzyme, this would suggest that the protease belongs to the serine alkaline protease family. For stability of the enzyme preliminary data with calcium has identified that calcium can stabilise the protease (as shown in
Cell Culture
The neuroblastoma cell line infected with the 22 L scrapie strain (N2a22L20) was used (Prior et al., (2007) J Virol 81: 11195-11207). The N2a22L20 cells were grown in DMEM medium supplemented with 10% FCS, 10 mM penicillin-streptomycin and 300 μg/ml geneticin. GT1 cells infected with the chandler scrapie isolate (GTK) and the 22 L scrapie strain (GT22L) (Nishida et al., (2000) J Virol 74: 320-325), were maintained in DMEM medium supplemented 5% FCS, 5% HS, 1 mM Sodium Pyruvate and penicillin-streptomycin. Cells were maintained at 37° C. in an atmosphere of 5% CO2 and 95% air. For standard lysis, cells were lysed in cold lysis buffer (LB) (0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 150 mM NaCl and 50 mM Tris-HCl (pH 7.5), 1 μg/ml pepstatin, 1 μg/ml leupeptin, 2 mM EDTA) and total protein concentration was measured using the BCA protein assay.
Western Blotting
For PrPSc analysis, lysates prepared to 50 μg were digested with 16 μg of proteinase K (pK)/mg of protein at 37° C. for 15 min, or as indicated, the reaction was then stopped with 1 mM pefabloc. Samples in loading buffer were boiled for 5 min and loaded onto 12% SDS-PAGE and analysed by western blotting employing standard techniques. For PrPC and or total PrP analysis, lysates were not pK digested. PrP was detected by incubating immunoblots with the antibodies indicated in the text followed by a horseradish peroxidase secondary antibody and developed by enhanced chemiluminescence (ECL).
PrPSc Degradation from Cells
For the non-cell assay, confluent cells were lysed into cold Lysis buffer (LB) lacking protease inhibitors, lysate was then adjusted to the protein concentration and pH indicated in the text. Enzyme (E) or heat denatured boiled enzyme (BE) was added to the lysate and the final buffer concentration in the lysate was 0.1M. Samples were incubated at 40° C. for the times indicated and were then solvent precipitated and examined for PrP levels.
Degradation of Prion in Brain Homogenate
Brain homogenate (10%, w/v), from Tga20 mice infected with the 22A scrapie strain and the BSE 301V strain, were prepared by passing the brain successively through 18, 20 and 23 gauge needles in PBS until free flowing. Infected brain homogenate was then resupsended in HEPES buffer pH 7.0, or in HEPES buffer pH 7.0 containing the units of E indicated or 1.53 units BE. BE samples were incubated for 15 min, E treated samples were incubated for the times indicated, in both cases the temperature of incubation is as stated in the text. Samples were then solvent precipitated, pellets were resuspended in LB buffer (0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 150 mM NaCl and 50 mM Tris-HCl (pH 7.5), 1ρg/ml pepstatin, 1ρg/ml leupeptin, 2 mM EDTA) and left on ice for 20 min. Where brain homogenate was pK (+) digested the homogenate was treated with 16 μg of proteinase K (pK)/mg protein for 30 min at 37° C. Samples were then analysed for PrP levels.
Degradation of PrPSc from Infected Cell Systems by Protease 16
Initial work on the ability of protease 16 to degrade the prion was carried out with the antibodies 11G5, 7A12, GE8, 8H4, that are directed to the carboxyl (C) terminus of the prion protein (as shown in
In case proteolysis resulted in just the loss of the 7A12 epitope or in the production of prion fragments that were not detected with 7A12, immunodetection was also carried out with the antibodies 8H4 (epitope 175-185 on Mouse PrP), 11G5 (epitope 115-130) and GE8 (epitope 183-191). No signal was obtained with any of these antibodies after the N2a22L20 lysate was digested with the protease 16 (E) (as shown in
Degradation of Prion Material from Infected Brain Homogenate by Protease 16
To profile the prion degradation ability of protease 16, its ability to degrade prion infected brain homogenate was examined. We chose mouse adapted scrapie strain 22A and BSE strain 301V, which had been passaged in Tga20 mice. Infected brain material was homogenised directly into HEPES buffer pH 7.0 and detergents were not added. Protease 16 degraded both 22A (as shown in
From studies on the resistance of prion strains to thermoinactivation 22A has been shown to be one of the more resistant scrapie strains. Its passage in mice of differing Sinc genotype has little effect on resistance properties. However, Sinc genotype significantly affects the resistance characteristics of 301V. Passage of 301V for example in C57BL mice (Prnpaa/SV) results in a BSE strain that is less thermoresistant than 22A/SV. In light of this, in terms of potential resistance properties, the profile of degradation of the protease was examined further with 22A. Although protease degradation of PrPSc from the cell lines did not result in the detection of prion fragments (as shown in
Finally, to enhance the ability to detect prion fragments, if fragments were produced after digestion with 1.53 units enzyme, the protein load was increased 10 fold (as shown in
The protease 16 was found to be optimally active above pH 8.0. Despite its alkaline nature it still retains activity at mild pH's and could breakdown PrPSc at pH 7.0. The degradation of PrPSc seen at this pH would relate to the protease itself, if the assay were carried out at pH11-12 the alkaline pH would have contributed to the loss in PrPSc seen. Nonetheless, the alkaline profile of this system could lend to the incorporation of this protease into detergent systems if required. In searching for a successful antiprion protease the challenge is to obtain an enzyme system, with the right substrate specificity, to breakdown the prion into non-infectious units. The experiments had successfully identified that protease 16 can degrade PrPSc, under mild conditions, to undetectable levels by western blot. The enzyme degraded both 22A and 301V from mouse brain homogenate at pH 7.0 within 15 min at both 40° C. and 22° C. This provides a strong indication that this enzyme has the potential to act as an environmental decontaminant.
Isolation and Purification of a Second Microbial Protease Capable of Degrading Prions.
Later studies also identified a second candidate protease, again identified from a Thermoactinomycete isolate 10 and this was denoted protease 10. Protease 10 was also later characterised as a Thermitase.
Characterisation of Protease 16
Protease 16 was cloned and heterologously expressed in a Lactococcus lactis host. The protease was later identified as a Thermitase and was found to maintain its ability to degrade the prion under mild conditions which would enable it ultimately to act as a mild prion decontaminant.
Cloning of the Thermoactinomyces sp 16 Protease
The Thermoactinomyces sp 16 enzyme responsible for the degradation of PrPSc under mild conditions was identified as a Thermitase (EC 3.4.21.66) after analysis by Mass spectrometry. The protease was then cloned from Thermoactinomyces sp 16 chromosomal DNA using a method adapted from Nikodinovic et al., (2003) Biotechniques 35:932-934, 936. Thermoactinomyces sp 16 was grown on nutrient agar containing 1% (w/v) starch, pH 7.0 at 55° C. A loop full of spores and mycelia were inoculated into 50 ml medium (g/l): soluble starch 30.0, bacto-peptone 5.0, yeast extract 5.0, CaCl22H2O, 0.5, MnCl24H2O 0.5, MgCl26H2O 0.48, KH2PO4 1.0, pHi 7.0. The culture was grown for 48 h at 55° C. and 200 rpm. 5% (v/v) of this culture was transferred to 50 ml production medium (g/l): maltose 30.0, bacto-tryptone 5.0, yeast extract 5.0, CaCl22H2O 0.5, MnCl24H2O 0.5, MgCl2 6H2O 0.48, KH2PO4 1.0, initial pH (pHi) 7.0. The cells were then grown at 40° C. and 200 rpm for 48 h. Cells were then removed from the culture by centrifugation at 10,000 g for 15 min. The pellet was dissolved in 10 ml of lysis buffer (25 mM Tris-HCl pH 7.5, 25 mM EDTA, 0.3 M sucrose, 50 μg/ml RNase A). To this 2 mg/ml Lysozyme and 1 mg/ml of Achromopeptidase were added and then incubated for 60 min at 37° C. at 200 rpm. 1% (w/v) SDS and 0.5 mg/ml Proteinase K were then added and incubated for 1.5 h at 55° C. at 200 rpm. 1/3 Vol of 5M NaCl was then added. The sample then underwent the Phenol/Chloroform/Isoamyl extraction twice, followed by isopropanol precipitation.
The gene encoding the Thermitase was PCR amplified using Thermitase FL-Sapl F and Thermitase FL-Xhol R as detailed in Table 5 below.
DNA Taq polymerase from Thermo scientific was used, whereas dNTPs were from Kapa Biosystems. The 1.16 kb product, using the restriction enzymes Sapl and Xhol was ligated to pAMJ2008, a high copy vector with its own signal peptide SP310mut2(9) and transformed into Escherichia coli DH5a. The full sequence was obtained through DNA sequencing. The Thermitase gene lacking the N-terminal signal peptide was generated by PCR amplification of the gene using the primer combination Thermitase ΔN-sapl F and Thermitase FL-Xhol R (see Table 2 above). This product was cloned into pAMJ2008, and transformed into E. coli DH5a in which the plasmid was developed. The entire plasmid containing the Thermitase gene lacking its signal peptide was subsequently cloned into L. lactis strain MG1363 generating L. lactis strain 16. Lactococcus lactis strain MG1363 and vector pAMJ2008 were both from Bioneer. The final sequence was confirmed by DNA sequencing.
Non Recombinant Protease Production
To produce the non-recombinant protease of Thermoactinomycete sp. 16, a standard inoculum was developed by transferring a loopful of spores and mycelia to 50 ml inoculum medium (g/l): soluble starch 30.0, bacto-peptone 5.0, yeast extract 5.0, CaCl22H2O, 0.5, MnCl24H2O 0.5, MgCl26H2O 0.48, KH2PO4 1.0, pHi 7.0. The culture was grown for 48 h at 55° C. and 200 rpm. 5% (v/v) standard inoculum was transferred to 50 ml production medium (g/l): maltose 30.0, bacto-tryptone 5.0, and yeast extract 5.0, CaCl22H2O 0.5, MnCl24H2O 0.5, MgCl2 6H2O 0.48, KH2PO4 1.0, pHi 7.0. The cells were then grown at 40° C. and 200 rpm for 72 h. Cells were then removed from the culture by centrifugation at 10,000 g for 15 min. The protease activity in the cell-free supernatant was then determined.
Recombinant Protease Production
To produce the recombinant protease of Thermoactinomycete sp. 16a standard inoculum of L. lactis stain 16 was developed by transferring 200 μl of frozen stock of L. lactis stain 16 (OD600 nm of 1) into 5 ml of production media (g/l): M17 broth 42.0, Glucose 10.0, Yeast Extract 20.0 and 1 ug/ml erythromycin. The culture was grown for 18 h at 30° C., and 200 rpm. 1 ml of inoculum (OD600 nm of 1) was transferred to 50 ml of production medium. The culture was grown for 8 h at 30° C., pH6.5 and 200 rpm. The culture was maintained at pH 6.5 by the addition of 3M KOH at identified time periods. Cells were then removed from the culture by centrifugation at 10,000 g for 15 min 4° C. The protease activity in the cell-free supernatant was then determined.
Purification of Protease
The protease of Thermoactinomycete sp 16 was purified from the cell-free supernatant in two steps. The first step was ammonium sulphate (0-60%) fractionation. To express the final ammonium sulphate concentration as % ammonium sulphate saturation the Green and Hughes calculations were used. The precipitate obtained after fractionation was suspended in 0.02M HEPES buffer, pH 7.0 and was dialysed against water for 24 h. The second step used was affinity chromatography on bacitracin-Sepharose 4B using a modification of the method of Stepanov and Rudenskaya. Bacitracin-Sepharose 4B was equilibrated with 0.04M Tris/HCL, pH 7.9. Enzyme solution in 0.04M Tris/HCL pH 7.9 was applied to the column and the column was washed with the same buffer to remove all unbound protein. In the case of the recombinant enzyme the enzyme required heat activation before being applied onto the column. It was heated at 50° C. for 20 min in 0.04M Tris/HCL pH 7.9, followed by 10 min at 60° C.). The protease was then eluted from the column with 25% (v/v) propan-2-ol in 0.04M Tris/HCL pH 7.9, containing 1M NaCl at a flow rate of 0.5 ml/min. The eluate was then dialysed.
Protease Assay
In the standard protease assay, activity was determined by the addition of 50 μl enzyme to 50 μl bovine serum albumin (BSA) (1%, w/v) in 0.1M CAPS buffer, pH 10.0 and incubated for 30 min at 40° C. or 60° C. (where indicated). The reaction was then stopped by the addition of 200 μl 5% (w/v) trichloroacetic acid (TCA). After 10 min the precipitate was centrifuged at 3,000 g for 10 min. Total protein concentration in the supernatant was measured using the bicinchoninic acid (BCA) protein assay kit (Sigma) or Bradford assay. A unit of activity is defined as the amount of enzyme releasing 1 mg TCA soluble protein fragments from BSA in 30 min at 40° C. or 60° C.
Western Blotting
For PrPSc analysis, lysates prepared to 50 μg were digested with 16 μg of proteinase K (pK)/mg of protein at 37° C. for 15 min, or as indicated, the reaction was then stopped with 1 mM pefabloc. Samples in loading buffer were boiled for 5 min and loaded onto 12% SDS-PAGE and analysed by western blotting employing standard techniques. For PrPC and or total PrP analysis, lysates were not pK digested. PrP was detected by incubating immunoblots with the antibodies indicated in the text followed by a horseradish peroxidase secondary antibody and developed by enhanced chemiluminescence (ECL).
Mice and RML Intracerebral Inoculations
CD1 male mice used and at 5-6 weeks of age they were injected with 20 μl of 1% RML brain homogenate intracerebrally into the left parietal region. Initial 10% brain homogenates was prepared in 0.32M sucrose, and was then diluted in 1% PBS containing 2% fetal bovine serum. Inoculated mice were monitored closely for clinical signs of mouse prion disease and were sacrificed at the point of development of neurological disease.
Degradation of Prion in Brain Homogenate
Brain homogenate (10%, w/v), from TgA20 mice infected with the RML scrapie strain was prepared by passing the brain successively through 18, 20 and 23 gauge needles in PBS until free flowing. 50 μg infected brain homogenate was resupsended in 0.1M HEPES buffer pH7.0 (B), or in 0.1M HEPES buffer pH7.0 containing the units of enzyme (E) indicated or boiled heat inactivate enzyme (BE). BE and E samples were incubated for 15 min, E treated samples were incubated for the time indicated, at 22° C. Samples were then solvent precipitated, pellets were resuspended in LB buffer (0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 150 mM NaCl and 50 mM Tris-HCl (pH 7.5), 1ρg/ml pepstatin, 1ρg/ml leupeptin, 2 mM EDTA) and left on ice for 20 min. Where brain homogenate was pK (+) digested the homogenate was treated with 16 μg of proteinase K (pK)/mg protein for 30 min at 37° C. Samples were then analysed for PrP levels.
Cloning of the Prion Degrading Protease of Thermoactinomycete p 16.
The purified serine protease of Thermoactinomyces sp 16 was analysed by Mass spectrometry, this identified the protease as a Thermitase. Jorgensen et al (12) recently published work (Jorgensen et al., (2013) Protein expression and purification 92:148-155) on the recombinant expression of an Actinomycete Laceyella sacchari Thermitase; their work identified that their full protease gene included a signal peptide, a pro peptide domain, followed by the mature protease domain. The propeptide domain enables correct folding of the mature protease, but is naturally removed by the host on secretion of the mature enzyme. Its removal is essential for enzyme activity as it inhibits the enzyme when bound. To isolate the protease primers were designed for the complete Thermoactinomyces sp 16 Thermitase gene (Table 6), from which the gene was isolated as a 1.16 kb PCR fragment. The gene was DNA sequenced (Table 6) and the translated protein sequence was identified to be composed of three distinct regions a 25 amino acid signal peptide, an 83 amino acid propeptide and the mature protease region of 279 amino acid (Table 7).
Using the GenBank BLAST P programme the pro-peptide was identified as possessing an 19 inhibitor (13) with complete homology to that of the L. sacchari Thermitase (12). The mature protease aligns with the Peptidase S8 family domain in Thermitase-like proteins; it differs from its most closely related Thermitase of L. sacchari by two amino acids as indicated in Table 7.
Recombinant Production of the Thermoactinomycete Sp 16 Thermitase
The protease gene lacking its signal peptide was cloned into the pAMJ2008 vector system, the PCR inserts including the cloning junctions were confirmed by DNA sequencing. The pAMJ2008 vector employs the L. lactis signal peptide SP310mut2 which facilitates secretion of the protease to the eternal milieu, as the signal peptide is cleaved during translocation. The expression strain chosen was the L. lactis strain MG1363 which itself lacks any host protease secretion. The Thermitase was released as an inactive pro-enzyme which required release of the prodomain, this was accomplished by heat treatment. The protease was produced in batch culture and was purified by ammonium sulphate (0-60%) fractionation, followed by affinity chromatography on bacitracin-Sepharose 4B (Table 8). The non-recombinant Thermoactinomycete sp 16 Thermitase was produced as described previously.
(L. lactis strain 16 was grown for 8 h at 30° C., pH6.5 and 200 rpm. Cells were then removed from the culture by centrifugation at 10,000 g for 15 min 4° C. The supernatant was then purified by (NH4)2SO4 (0-60%) fractionation, heat treatment followed by affinity chromatography on Bacitricin-Sepharose 4B).
Degradation of PrPSc from Infected Brain Homogenate by the Thermoactinomycete Sp 16 Thermitase.
It was decided to recombinantly produce the Thermitase of the Thermoactinomycete sp 16 and assess whether it would maintain its ability to degrade PrPSc under mild conditions at 22° C. and at pH 7.0 after 15 min. Prion degradation was tested with the scrapie strain RML. 50 μg RML brain homogenate was exposed to 0.5 units/ml of recombinant Thermitase (RE) and of the original enzyme (E) at 22° C. for 15 min, this was found to be sufficient to remove all traces of the RML prion strain as detected by western blot (as shown in
Results of Cloning of the Thermoactinomyces sp 16 Protease
The Thermitase was successfully expressed in the L. lactis strain MG1363 under the control of the vector system pAMJ2008. This vector has previously been reported to successfully allow for the expression of the Thermitase of L. sacchari in L. lacti. The vector's SP310mut2 facilitated the external secretion of our protease which then required heat to enable activation due to its prodomain. As mentioned the Thermoactinomycete sp16 Thermitase is composed of a pro-domain, these domains are known for their significance in the correct folding of specific active mature proteases, but they also render the protease inactive whilst attached. In the case of the prodomain of subtilisin it inhibits activity through the binding of its prodomain C-terminus to the mature enzymes active site much like a substrate and can be released auto-catalytically under appropriate conditions. Like other Thermitases heating facilitated activation and release of the mature Thermoactinomycete sp16 Thermitase.
The recombinant Thermitase 16 was tested for its ability to degrade the RML prion, and like the original enzyme it degraded the prion within 15 min at 22° C. and at pH 7.0. Thus far this protease is believed to be the first to degrade prion material under such mild conditions. The Thermitase 16 represents the first enzyme capable of degrading the prion under true environmental conditions. The nature of the Thermitase enables for diversity in the cleavage sites of its substrate. They belong to the subtilases, and possess good substrate diversity this characteristic has been seen as beneficial in the degradation of proteins in to smaller peptides than potentially seen with subtilisin itself. This diversity in the degradation profile of this group of proteases is most likely the reason why Thermitase 16 can overcome the complex protein β-sheet structure of PrPSc which has challenged the heretofore methods employed to decontaminate prion material.
Effect of pH on Protease Activity of the Original and Recombinant Protease 16
The original and recombinant proteinase 16 were assayed using BSA (1%, w/v) as substrate in 0.1M Universal buffer at the pH values indicated for 30 min at 40 C.
Cloning of the Thermoactinomyces sp 10 Protease
It was decided to clone and analyse protease 10 (Thermitase 10) which had also been found to degrade prions under mild conditions at pH 7.0 and 22° C. The protease sequence differs significantly to those published, also to the sequence of protease 16 which is described above. Similar methods were employed as for the cloning and analysis of protease 16.
Cloning of the Thermoactinomyces sp 10 Protease
The Thermoactinomyces sp 10 enzyme responsible for the degradation of PrPSc was cloned using the primers employed for Thermoactinomyces sp 16. Cloning was carried out as preformed for Thermoactinomyces sp 16. The gene encoding Thermitase 10 was PCR amplified using Thermitase FL-Sapl F and Thermitase FL-Xhol R (as detailed in table 5) and its product was ligated to pAMJ2008 and transformed into Escherichia coli DH5α.
The full sequence was obtained through DNA sequencing. The Thermitase gene lacking the N-terminal signal peptide was generated by PCR amplification of the gene using the primer combination Thermitase ΔN-sapl F and Thermitase FL-Xhol R (as detailed in Table 5). This product was cloned into pAMJ2008, and transformed into E. coli DH5α in which the plasmid was developed. The entire plasmid containing the Thermitase gene lacking its signal peptide was subsequently cloned into L. lactis strain MG1363 generating L. lactis strain 10. The final sequence was confirmed by DNA sequencing.
Recombinant Protease Production
The recombinant production of the protease of Thermoactinomycete sp. 10 was produced in the same manner as that of Thermoactinomycete sp. 16.
Cloning of the Prion Degrading Protease of Thermoactinomycete Sp 10
The Thermitase of Thermoactinomyces sp 10 was cloned using the same cloning strategy used for Thermoactinomyces sp 16. The gene was DNA sequenced and is detailed in Table 9 and the translated protein sequence was identified to be composed of three distinct regions a 25 amino acid signal peptide, an 83 amino acid propeptide and the mature protease region of 279 amino acid and is detailed in Table 10, as per Thermoactinomycete 16.
The protease gene sequence differed from both the Thermitase of the published sequence of L. sacchari and that of Thermoactinomyces sp 16. The bases that differ from both are highlighted in yellow, and those highlighted in red are specific differences between the gene of protease 10 and that of L. sacchari. As with the Thermitase of Thermoactinomyces sp 16, Thermitase 10 aligns with the Peptidase S8 family domain in Thermitase-like proteins. It differs from its most closely related published Thermitase of L. sacchari by 12 amino acids. From Thermitase 16 it differs by 9 amino acids, one of which is located in the prodomain of the protease (Table 10).
The Thermitase of Thermoactinomyces sp. 10 was expressed in the L. lactis strain MG1363 under the control of the vector system pAMJ2008, as per the Thermoactinomyces sp 16 Thermitase. Like the Thermoactinomyces sp16 Thermitase, Thermitase 10 has a pro-domain, a domain that is known for its significance in the correct folding of specific active mature proteases, it also renders the protease inactive whilst attached. This applied to Thermitase 10, and the protease required heating to enable activation of the mature protease.
As shown
The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.
Indications of Deposited Biological Material
A deposition of biological material was made to National Collections of Industrial and Marine Bacteria Limited (NCIMB) for the purposes of filing one or more patent applications. The National Collections of Industrial and Marine Bacteria Limited (NCIMB) is a recognised International Depository Authority (IDA) under the Budapest Treaty and the deposition of biological material was made on the same terms as those laid down in the Treaty. The deposit has been assigned a number along with the prefix “NCIMB”.
The deposited biological referred to in this application is as follows:
Thermoactinomyces sp 16 strain
| Number | Date | Country | Kind |
|---|---|---|---|
| 1522814 | Dec 2015 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/082557 | 12/23/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/109170 | 6/29/2017 | WO | A |
| Number | Date | Country |
|---|---|---|
| 9859045 | Dec 1998 | WO |
| WO 9859045 | Dec 1998 | WO |
| 2008057293 | May 2008 | WO |
| Entry |
|---|
| Meloun et al., “Complete primary structure of thermitase from Thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteinases”, FEBS 2463 vol. 183 No. 2 pp. 195-200 (Year: 1985). |
| Jorgensen et al.,“Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis”, Protein Expression and Purification 92: 148-155 (Year: 2013). |
| Jorgensen et al. Thermitase amino acid sequence ; GenBank Deposit ADL09141.1; downloaded Nov. 6, 2021. (Year: 2021). |
| Meloun et al.: “Complete primary structure of thermitase from thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteinases” FEBS Letters 1985 NL, vol. 183, No. 2, 1985, pp. 195-200. |
| Verma et al.: “Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.”. Protoplasma May 2014. vol. 251. No. 3. May 2014 (May 2014). pp. 711-718. |
| PCT/EP2016/082557 International Search Report dated Mar. 28, 2017. |
| Muller-Hellwig et al.: “Biochemical evidence for the proteolytic degradation of infectious prion protein PrPsc in hamster brain homogenates by foodborne bacteria.”.Systematic And Applied Microbiology Mar. 2006. vol. 29. No. 2. Mar. 2006 (Mar. 2006). pp. 165-171. |
| Number | Date | Country | |
|---|---|---|---|
| 20200370030 A1 | Nov 2020 | US |
