WO2017125886A1

WO2017125886A1 - Use of slpa protein from deinococcus radiodurans as a screen against ultraviolet radiation

Info

Publication number: WO2017125886A1
Application number: PCT/IB2017/050304
Authority: WO
Inventors: Dario PIANO
Original assignee: Università Degli Studi Di Cagliari
Priority date: 2016-01-22
Filing date: 2017-01-20
Publication date: 2017-07-27
Also published as: ITUB20160035A1

Abstract

The present invention relates to the use of the SlpA protein, isolated from the S-layer of Deinococcus radiodurans, as a screen against ultraviolet radiation. In particular, the present invention relates to the use of said SlpA protein, in association with its cofactor, i.e. the carotenoid deinoxantin, as a screen against ultraviolet radiation.

Description

USE OF SlpA PROTEIN FROM DEINOCOCCUS RADIOD URANS AS A SCREEN AGAINST ULTRAVIOLET RADIATION

DESCRIPTION

Technical field

The present invention relates to the use of the SlpA protein (or DR 2577), isolated from the surface layer (or S-layer, as it will hereinafter be called for simplicity) of the bacterium Deinococcus radiodurans, as a protective screen against ultraviolet radiation.

In particular, the present invention relates to the use of said SlpA protein, in association with its cofactor, the carotenoid deinoxantin, as a protective screen against ultraviolet radiation.

Furthermore, the present invention also relates to the extraction of deinoxantin from its association with the SlpA protein in preparative-scale amounts.

Background art

The bacterium Deinococcus radiodurans is a pink-coloured bacterium which is well known in the literature for, among other things, its ability to withstand a broad range of extreme conditions. Merely by way of example, it can withstand quite well large doses of ionizing radiation and ultraviolet radiation (UV), especially in exsiccated conditions. It is therefore important to widen the knowledge of the properties and possible applications of the same and, most importantly, of the components thereof. The present Author has found out that, as for many other bacterial species (such as, for example, Thermus thermophilus, Haloferax volcanii, Synechocystis, Bacillus cereus, Bacillus subtilis, Clostridium difficile, Aquaspirillum serpens), also the external surface of this bacterium is coated with a prevalently protein-based paracrystalline S- layer, wherein the DR 2577 protein, also referred to as "Surface Layer Protein A" (or SlpA, as it will hereinafter be called for simplicity), is one of the main components. Apart from the notorious self-assembly capability even at low concentrations, at present not all functions and action mechanisms of this protein are well-known. At any rate, due to its structural and functional characteristics, it has been demonstrated that the protein may be involved in the protection mechanisms of the bacterium itself. Likewise, the present Author has found out that the carotenoid deinoxantin that is present in the S-layer is associated with the SlpA protein and is bound thereto via non- covalent interactions, thus giving the bacterium its characteristic pink colour. Carotenoids are a known group of natural pigments with antioxidizing properties and with functions ranging from protection against reactive oxygen species (ROS) in oxidative stress conditions to photoprotection correlated to photooxidation in photosynthetic organisms. Consequently, deinoxantin also appears to be potentially involved in the protection mechanisms of the bacterium.

Until now, it had not been clarified whether the SlpA protein, as such or in native form, i.e. in association with its cofactor, i.e. the carotenoid deinoxantin, is involved or not in the ability of the bacterium Deinococcus radiodurans to withstand large doses of ultraviolet radiation (UV), especially in exsiccation conditions.

In many technical application fields there is an increasing demand for substances, associations and compositions offering at least good and versatile capability of effectively and stably filtering and/or absorbing ultraviolet radiation.

By way of non-limiting example, in dermatology it would be useful to have available dermatologic and/or cosmetic products capable of protecting the skin effectively and for a long time against the damages causes by exposition to ultraviolet rays. The products currently available, in fact, which are generally based on protective filters consisting of small screening molecules (also synthetic ones) that in many cases are not very durable, have not turned out to be fully satisfactory for all phototyes.

Furthermore, in biotechnology applications it would be useful to rely on an effective system for ultraviolet radiation absorption combined with fluorescence emission, which could be used as a marker in fluorescence microscopy.

Likewise, it would be useful to have available a system with UV filter properties, associated with self-assembly characteristics, for creating long-lasting filtering films with nanometric structural regularity, which could be used for aerospace and/or building engineering applications (e.g. production of more efficient and less wearable solar panels or reflecting surfaces).

Technical Problem

Those skilled in the art strongly feel the need for having readily available substances suitable for the production of versatile, resistant and durable UV filters, so as to be able to satisfactorily solve the problems highlighted above. The object of the present invention, therefore, is to adequately find a solution to the above-described technical problem.

Summary of the Invention

The Applicant has now found that a suitable composition comprising the SlpA protein, preferably the SlpA protein in association with its carotenoid cofactor deinoxantin from Deinococcus radiodurans, can adequately solve the technical problem highlighted above.

One object of the present invention is, therefore, the use of said SlpA protein, preferably of said protein in association with deinoxantin from Deinococcus radiodurans, for the preparation of a UV filter as set out in the appended independent claim.

It is another object of the present invention to provide a composition comprising said substance(s) for the above-mentioned use, as set out in the appended claims.

It is a further object of the present invention to provide a method for the production of deinoxantin, as set out in the appended claims.

Some preferred embodiments of the present invention are set out in the appended dependent claims.

The preferred embodiments of the present invention illustrated in the following description only have an explanatory purpose and are by no means intended to limit the application scope of the present invention, which will be immediately apparent to those skilled in the art.

Detailed Description of the Invention

The present invention relates to the use of the SlpA protein, preferably of the SlpA protein in association with deinoxantin from Deinococcus radiodurans, as a filter against ultraviolet radiation.

The present invention further relates to the use of the SlpA protein, preferably of the SlpA protein in association with deinoxantin from Deinococcus radiodurans, for the preparation of a composition/product having properties as a protective/absorbent filter against ultraviolet radiation.

In one embodiment of the invention, said protein is extracted from the S-layer of the bacterium Deinococcus radiodurans.

In another embodiment of the invention, said protein extracted from the S-layer of Deinococcus radiodurans is in association with its cofactor deinoxantin.

In one embodiment of the present invention, the SlpA protein in its native form, i.e. in association with its cofactor deinoxantin, is prepared by means of a method for selectively isolating the protein and its cofactor in solution and in pure form, said method schematically comprising at least the following first two steps:

1) production, in suspension, of homogeneous fragments of the bacterial cellular wall of Deinococcus radiodurans;

2) isolation, in solution, of the SlpA protein and its cofactor deinoxantin by cold extrusion of the fragments of bacterial cellular wall obtained in step 1);

3) optionally, concentration and/or modulation of the assembly capability of the SlpA in solution obtained in step 2) by adding an efficacious quantity of a mild detergent;

4) optionally, purification of SlpA and its cofactor via a preparative chromatographic method;

wherein the whole method is carried out at a low temperature comprised, on average between 2°C and 10°C or between 2°C and 6°C, preferably between 3°C and 5°C, more preferably at approximately 4°C, even more preferably at 4°C.

Said method may possibly comprise a fifth step, in which the deinoxantin is separated or extracted from the SlpA protein by means of a suitable polar or apolar solvent for the purpose of obtaining the oxidized (orange form) or reduced (pink form) variants of deinoxantin to give pure deinoxantin.

Step 1) is carried out in accordance with the teaching of Farci D. et al. (2014), New features of the cell wall of the radio-resistant bacterium Deinococcus radiodurans, Biochim. Biophys. Acta 1838, 1978-1984), which is wholly incorporated herein as a reference.

In practice, the cells of the bacterial culture (produced as described above) are collected by centrifugation (5,000 rpm x 5 min), followed by suspension of the bacterial pellet thus obtained in 50mM phosphate buffer at pH 7.8 (buffer A); the cell suspension thus obtained is lysated by means of three French Press fragmentation cycles for cell expansion according to known standard procedures (three cycles at 1,100 psi) (see supra Farci D.); the lysated cell suspension is then subjected to multiple centrifugations to remove the roughest residual fractions, i.e. the residual fraction of non-lysated cells (first centrifugation at 5,000 rpm x 10 min), the larger cellular wall fragments (second centrifugation at 5,000 rpm x 10 min), the smaller cellular wall fragments (third centrifugation at 48,000 rpm x 10 min); the resulting pellet is then re- suspended in buffer A in the presence of 15mM EDTA at pH 7.4 and washed (centrifugation at 48,000 rpm x 10 min), and is subsequently digested in the presence of lysozyme to remove surface polysaccharides and finally washed several times to remove the lysozyme as well, after having been re-suspended in buffer A (centrifugation at 48,000 rpm x 10 min), to give a homogeneous suspension of wall fragments.

In step 2), the homogenous suspension of wall fragments obtained in step 1 is subjected to a cold extrusion process (cold compression/expansion by means of at least one French Press cycle at 1,100 psi), which disassembles the protein together with its cofactor from the wall fragments by expansion until they pass in solution. The residual membrane fragments and those fractions which are still insoluble are eliminated by centrifugation (48,000 rpm x 10 min), and the liquid phase (supernatant) is recovered. Since the native protein obtained in solution in step 2) has the tendency to self- assemble depending on its concentration, in the next step 3) this tendency is removed, if necessary, or at least reduced to an acceptable level for the subsequent processing steps. For example, the native protein solution obtained in step 2) is supplemented with a suitable quantity of a mild, non-ionic detergent capable of increasing the solubilization constant and decreasing the nucleation index of the proteins, thereby allowing the proteins to start self-assembling in the form of regular sheets at concentrations higher than the normal ones. For example, said quantity is in the range of 0.01% to 0.1%) in weight, preferably 0.03%> to 0.09% in weight, more preferably 0.04% to 0.08% in weight, even more preferably 0.05% to 0.07% in weight, and even more preferably of approximately 0.06% in weight, with reference to 100 mL of protein solution.

Said mild detergent is preferably selected from the group of alkyl maltosides and glucosides, such as, for example, dodecyl-maltoside, in any alpha or beta anomeric form thereof.

In these conditions, the protein/cofactor complex in solution can be concentrated, for example, by ultrafiltration by using polyethersulfone membranes, up to values of approx. 5-6 mg/mL, or even higher, so that large amounts of product can be purified. In the optional next step 4), purification of the protein/cofactor complex is achieved by using traditional chromatographic methods, e.g. Size Exclusion Chromatography (SEC), as will be described in the test section, to give the pure native protein.

The above-described method can be successfully applied on a laboratory scale, on a small scale and also on a semi-industrial or industrial scale, by appropriately adapting the equipment to the required product amounts.

For example, it will be possible to switch from discontinuous-flow French Press equipment for laboratory and/or small-scale preparations to continuous-flow French Press equipment and bioreactors for producing the Sip A protein and its cofactor deinoxantin on an industrial scale.

This method has proved to be particularly useful also for producing large amounts of deinoxantin.

De facto, deinoxantin is known as one of the most powerful carotenoids in terms of antioxidizing activity, in addition to having a number of other peculiar properties. Carotenoids in general represent an important class of molecules that can be used, for example, as food supplements for preventing oxidative stress, or as drugs in the therapy for several degenerative diseases. This means that new sources of carotenoids are essential and strongly desired in an increasingly growing market. Thanks to the easy and delicate way of isolating the native Slpa protein with high yield and purity, the method just described also provides a simple and low-cost source of deinoxantin. In fact, the native SlpA protein in solution obtained in the above step 2) or, preferably, in the above step 4), was precipitated with PE8000 in phosphate buffer. The resulting pellet was exsiccated and then extracted with pure solvents, respectively polar ones (methanol, ethanol and acetone) to give an orange-coloured form or deinoxantin, or apolar ones (chloroform and hexane) to give a pink-coloured form of deinoxantin. The following test section will illustrate more in detail, but still merely by way of non- limiting example, one preferred procedure for preparing native SlpA protein and deinoxantin from Deinococcus radiodurans in accordance with the present invention.

Test procedure - Method for isolating, in solution and in pure form, native SlpA protein with its cofactor deinoxantin from the bacterial strain Deinococcus radiodurans The bacterial strain of Deinococcus radiodurans Rl (ATCC 13939) was cultivated, according to the above-mentioned literature, in Tryptone/Glucose/Yeast (TGY) culture broth (all three components were supplied by Microbiol Diagnostici, Italy) for 24 hr at 30°C, under stirring at 250 rpm. The resulting bacterial cells were collected by centrifuging 0.75L cultures (4°C, 5,000 rpm x 10 min) and re-suspended in 50mM sodium phosphate at pH 7.8 (buffer A). One pellet of bacterial cells of approx. 3-4 g/L of culture was collected on average. These yields remained almost unchanged when the cultures were carried out in volumes of 0.75 L to 6 L, i.e. up to a yield of approx. 18-24 g. The bacterial pellet suspension was then treated with DNasi and subjected to cellular lysis in a step of sudden pressure/expansion by using either a discontinuous French Press system, in a first case of small-scale production, or a continuous French Press system, in a second case of production on a larger scale. The residual non- lysated cells and the larger fragments were removed by low-speed differential centrifugation (4°C, 2 x 2,000 rpm x 10 min). The final supernatant was subjected again to centrifugation (4°C, 48,000 rpm x 10 min) and the resulting pink pellet (colour due to deinoxantin) was re-suspended in phosphate buffer A. In order to increase the quantity of bacterial cells collected, 6-8 L of cultures were grown in a suitably sized fermentation bioreactor, thereby increasing the quantity of collected cells up to 60-90 g (instead of the expected 24-32 g). The greater quantity of cells collected made it necessary to increase the final re-suspension volume significantly, and this fact also made it necessary to switch from a discontinuous French Press system (from which approx. 500 mg of cellular wall/L of culture medium could be obtained, i.e. approx. 4 g of cells) to a similar system of the continuous type (from which approx. 700 mg of cellular wall/L could be obtained, i.e. approx. 10 g of cells), with a slightly lower extraction yield but through a simpler procedure that can be easily scaled up to the desired dimension. In this manner (i.e. through the use of a suitably sized bioreactor and an appropriate continuous French Press cell disruption system), it was possible to scale the above-described process from a small scale up to a larger scale for larger quantities, which could be modulated up to an industrial scale. In order to remove the surface polysaccharides, the suspension of cellular wall fragments was first incubated under stirring (at 800 rpm) with 100 μg/mL of lysozyme (thus weakening the peptidoglycane) for 8 hr at 30°C. Then, for the purpose of obtaining the native SlpA protein in solution, a fragmentation (or component disassembly) step was carried out by subjecting the above suspension of cellular wall fragments to a cold extrusion step characterized by pressurization (at 1, 100 psi) followed by extrusion at low temperature (4°C), as opposed to a normal expansion that would take place in hollow bodies such as intact cells (when using a discontinuous or continuous French Press system according to the quantity of product to be treated); this cold extrusion step was then followed by centrifugation (4°C, 48,000 rpm x 10 min) and recovery of the clear supernatant containing the protein component in solution.

The application of the cold extrusion step of the present invention to the cellular wall fragments after digestion with lysozyme by using discontinuous and continuous French Press systems led to release of the native SlpA protein in association with its cofactor deinoxantin, in solution, with very high efficiency, so that 3-4 g of bacterial cell pellet (1L cultures, discontinuous French Press) typically allowed the isolation of approx. 68 mg of SlpA from approx. 430-570 mg of cellular wall fragments, while for large-scale preparations (6-8L culture in bioreactor, continuous French Press) allowed the isolation of approx. 0.65-0.85 g of native SlpA.

The native protein sample obtained after this solubilization step was purified by loading it onto a 20mL size exclusion chromatography column (Superose 6 10/300GL, GE Healthcare) previously equilibrated in buffer (50mM sodium phosphate at pH 7.4, 0.06% (w/v) of β-DDM) at a flow velocity of 0.3 mL/min.

The purity of the sample was analyzed by native (BN-PAGE) and denaturing (SDS- PAGE) protein electrophoresis.

In order to isolate/extract deinoxantin from SlpA, the native SlpA in solution obtained in the previous chromatographic purification step was preferably precipitated by centrifugation (4°C, 4,000 rpm x 30 min) with PEG8000 at 10% weight/volume in 50mM sodium phosphate buffer at pH 7.4. After centrifugation, the supernatant was eliminated and the resulting pellet was exsiccated at 4°C in no-frost conditions and under constant ventilation for 6 hr. Finally, deinoxantin was extracted from said pellet by means of pure solvents to give:

- an orange (oxidized) form, when extracted by using a polar solvent (methanol, ethanol and acetone); - a pink (non-oxidized or reduced) form, when extracted by using an apolar solvent (chloroform and hexane).

The same extraction method can be applied to the native SlpA protein in solution prior to chromatographic purification, although the final product thus obtained will be less pure.

In order to verify if the SlpA protein and its cofactor deinoxantin are involved in the resistance to UV radiation which is characteristic of Deinococcus radiodurans, the Author cultivated both the native type of SlpA and its deletion mutant (ASlpA), and compared the growth thereof in the absence and in the presence of UVC radiation. In normal conditions, the growth of the mutant turned out to be slightly less than that of the native type, and this tendency was observed to be much more pronounced when the same tests were conducted in the presence of UVC radiation (the mutant turned out to be much more sensitive to UVC light, i.e. unprotected against it). Similar experiments, conducted by pre-exposing dry cells of both strains (SlpA and ASlpA) to UVC radiation, showed a strong inhibition of the mutant's growth. These results clearly indicate that SlpA, in association with deinoxantin, plays a role as a guard/filter against ultraviolet radiation, especially in (extreme) exsiccation conditions.

Consequently, the SlpA protein, preferably in association with its carotenoid cofactor deinoxantin, can advantageously be used for the preparation of compositions, formulations, products, devices acting as a filter against UV radiation for a number of technological applications.

By way of example, which by no means should be considered to limit the broad application potential of the invention, the SlpA protein, preferably the SlpA protein in association with its cofactor deinoxantin from Deinococcus radiodurans, can be used for the preparation of dermatologic and/or cosmetic compositions/formulations (e.g. in the form of a cream, a gel, an oleogel, a lipogel, a paste or the like) having different cutaneous properties, e.g. acting as a solar filter with exceptional stability and resistance to UV radiations.

Merely by way of example, the SlpA protein, preferably the SlpA protein in association with its cofactor deinoxantin from Deinococcus radiodurans, is present in the dermatologic and/or cosmetic composition in a quantity comprised between 0.01 and 5 mg/mL of composition, preferably between 0.05 and 2.5 mg/mL of composition, more preferably between 0.1 and 1 mg/mL of composition. The remaining portion of said compositions usually consists of a base comprising an efficacious quantity of one or more active ingredients and/or an appropriate number of conventional excipients, diluents, additives, absorption promoters, adjuvants, filling agents/fillers, stabilizers, preservatives and carriers in well-known variable mutual ratios as commonly used by those skilled in the pharmaceutical art, depending on the desired type of composition and application.

Furthermore, also due to its self-assembly properties, the SlpA protein, preferably the complex of SlpA protein with deinoxantin from Deinococcus radiodurans, can advantageously be used for making UV radiation filtering films with nanometric regularity to be applied to surfaces of various kinds, e.g. for aerospace and building engineering applications, e.g. for the production of solar panels or reflecting surfaces. It has also been found that the SlpA protein and the complex thereof with deinoxantin have peculiar spectroscopic properties, in particular properties of absorption in the ultraviolet range and of fluorescence emission in the visible range. Advantageously, these characteristics make SlpA, preferably the complex thereof with deinoxantin, usable for the preparation of biomarkers for fluorescence microscopy.

Industrial Applicability

The present invention provides the use of the SlpA protein, preferably of the complex thereof with deinoxantin from Deinococcus radiodurans, as an efficient and stable filter resistant to ultraviolet radiation, for the preparation of compositions, formulations, products, devices having filtering properties against UV radiation for a number of technological applications.

In particular, the present invention provides the use of the SlpA protein, preferably of the complex thereof with deinoxantin from Deinococcus radiodurans, as a solar filter with stability and resistance to UV radiations, for the preparation of dermatologic and/or cosmetic compositions.

The present invention also provides a method for large-scale, low-cost production of the carotenoid deinoxantin.

Claims

1. Use of the SlpA protein and/or of the SlpA protein in association with deinoxantin from Deinococcus radiodurans as a filter against ultraviolet radiation.

2. The use of the SlpA protein and/or of the SlpA protein in association with deinoxantin from Deinococcus radiodurans according to claim 1, for the preparation of a composition and/or a formulation and/or a product and/or a device having properties as a stable and long-lasting protective and/or absorbent filter against ultraviolet radiation.

3. The use of the SlpA protein and/or of the SlpA protein in association with deinoxantin from Deinococcus radiodurans according to claim 1 or 2, for the preparation of a dermatologic and/or cosmetic composition, such as a solar filter having exceptional stability and resistance to UV radiations.

4. The use according to claim 3, wherein said composition is in the form of a cream, a gel, an oleogel, a lipogel, a paste, and said SlpA protein and/or said SlpA protein in association with deinoxantin from Deinococcus radiodurans is present in an efficacious quantity comprised between 0.01 and 5 mg/mL of composition.

5. The use of the SlpA protein and/or of the SlpA protein in association with deinoxantin from Deinococcus radiodurans according to claim 1 or 2, for the preparation of a UV-radiation filtering film with nanometric structural regularity for aerospace and building engineering applications, for the production of solar panels or reflecting surfaces.

6. The use of the SlpA protein and/or of the SlpA protein in association with deinoxantin from Deinococcus radiodurans according to claim 1 or 2, for the preparation of a biomarker for fluorescence microscopy.

7. A composition and/or a formulation and/or a product and/or a device comprising an efficacious quantity of SlpA protein and/or of SlpA protein in association with deinoxantin from Deinococcus radiodurans, for use as a stable and long-lasting protective and/or absorbent filter against ultraviolet radiation.

8. A method for extracting the carotenoid deinoxantin from the SlpA protein- deinoxantin from Deinococcus radiodurans complex, said method comprising;

- precipitating the native SlpA protein in solution, obtained after SEC chromatographic purification, by centrifugation (at 4°C, 4,000 rpm for 30 min) with PEG8000 at 10% weight/volume in 50 mM sodium phosphate buffer at pH 7.4;

- removing the supernatant and exsiccating the resulting pellet for 6 hours;

- extracting the deinoxantin from said exsiccated pellet by means of pure solvents to give:

- an orange (oxidized) form of the same, when extracted by using a polar solvent, methanol, ethanol, acetone;

- a pink (non-oxidized) form of the same, when extracted by using an apolar solvent, chloroform and hexane.