Pernisine is extracellular subtilisin-like proteinase from yperthermophilic archaeon Aeropyrum pernix. Extraordinary stability at high temperatures and presence of denaturants makes pernisine suitable for industrial pplications, because of its ability to degrade infective prion protein aggregates. Pernisine is homologous to the intensively studied Tk-subtilisin from Thermococcus kodakarensis, which contains three insertion sequences and six additional Ca2+ binding sites compared to mesophilic subtilisins. These insertion sequences, together with additional Ca2+ binding sites, contribute to folding and high thermostability of Tk-subtilisin. All Tk-subtilisin insertions and calcium binding sites are conserved in pernisine. However, pernisine contains an additional insertion sequence, absent in other subtilisin-like proteases characterized to date. Here we investigated the role of this unique insertion sequence in pernisine. This insertion consists of eight amino-acid residues and is located at the N-terminus of pernisine catalytic domain. The three residues of this insertion (D134, V135 and N136) form conserved calcium-binding motif DX[D/N]XDG, which constitutes an additional surface loop, as predicted in the 3D model of pernisine. Using spectrofluorimetry, circular dichroism and polyacrylamide gel electrophoresis, we have shown that compared to the wild-type pernisine, it's variant with deleted insertion sequence requires higher concentration of calcium ions to initiate conformational transition from unfolded to folded state of pernisine. Furthermore, pernisine lacking this insertion shows lower activity and thermal stability than its wild-type variant. Together, our results indicate that this novel insertion is an additional Ca2+ binding site, contributing to the adaptation of pernisine to the extreme environment of its host organism A. pernix.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 5168760Prions (PrPSc), a pathological form of a cellular prion protein (PrPc ), are responsible for transmissible spongiform encephalopathies. In contrast to bacteria and viruses, prions are extremely resistant to all kinds of decontamination practices, and are capable of binding tightly to surfaces, including those made of stainless steel, where they can persist and retain infectivity for a very long time. Our aim was to develop an effective decontamination procedure based on enzymes produced in a bacterial heterologous expression system that would be applicable also on sensitive and expensive medical instruments and equipment. Materials and Methods: For this purpose, we tested pernisine, a thermostable serine protease produced by an extremophilic archaeon, Aeropyrum pernix, and several recombinant varieties that were produced in Escherichia coli and S. rimosus expression systems. Activity of these enzymes was tested by western blot using normal bovine brain for PrPc degradation and bovine brain infected with bovine spongiform encephalopathy (BSE) for PrPSc degradation. Pernisine completely degraded PrPc and PrPSc, while recombinant varieties gave various results. Two varieties degraded PrPc and PrPSc, but it was necessary to optimize the enzyme to substrate ratio to obtain an appropriate effect. It was necessary to use around three times higher concentration of the recombinant variety to obtain activity comparable with that of the native pernisine. Preliminary results show that pernisine has a potential as an effective decontamination product and some recombinant varieties gave promising results, but further work will be required to develop an enzymatic system that would be equally effective and suitable for industrial application.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 4761466At the Congress FEBS3 +, I was a member of organizing committee. We presented our results obtained for pernisine, produced in Streptomyces. Recombinant pernisine was active, it was capable to degrade infective prions. We reported on the process of express, isolation, cleansing and the activity of pernisine.
C.07 Other editorial board
COBISS.SI-ID: 4943992Work was awarded by Student Faculty "Prešernova nagrada." Pernisine is an extracellular serine protease from the hyperthermophilic archaea Aeropyrum pernix. Development of an efficient recombinant expression system is crucial for development of industrial applications and further characterisation of this enzyme. Several expression vectors for overexpression in E. coli were constructed based on pMCSG7 and pMD204 vectors and codon-optimised pernisine gene. Initial problems with pernisine purification were resolved with translocation of enzyme accumulation into periplasm. Enzyme yield of ~4 mg L-1 production culture was achieved with isolation from the periplasmic fraction and purification of His-tagged enzyme with Ni-NTA column affinity chromatography. Pernisine is matured into an activate form with autocatalytic cleavage of proregion from the N-terminus. Based on the predicted cleavage sites of post-translational maturation unprocessed pernisine (Prn1), enzyme without signal sequence (Prn26) and maturated enzyme without the proregion (Prn92; Prn94) were synthesised. Zymography confirmed successful folding of all recombinant pernisines into an active proteolytic conformation. CD-spectrometry and intrinsic fluorescence emission spectrometry were used to demonstrate high thermal stability of recombinant pernisine and high proteolytic activity at 100 °C was shoved with azocasein assays. We have demonstrated that auto-proteolysis of pernisine samples can be problematic during enzyme production and biochemical characterisation. Binding of calcium ions (Ca2+) results in conformational changes in structure, probably via formation of Ca2+-binding loop. This increases conformational stability and proteolytic activity, thus consequently also the rate of autocatalytic maturation and auto-proteolysis in pernisine solutions.
E.01 National awards
COBISS.SI-ID: 8993657I was invited speaker at Conference organized by Serbian Biochemical Society in Belgrade as a representative of Slovenian Biochemical Society.
B.04 Guest lecture
COBISS.SI-ID: 5122680