Method for coating substrates with at least one monolayer of self-assembling proteins

Abstract

The invention relates to methods for coating a substrate with at least one monolayer of self-assembling proteins, using stabilized aqueous solutions with self-assembling proteins, and also to substrates obtainable as a result. Methods for stabilizing solutions with self-assembling proteins, and the stabilized solutions obtainable therefrom, are likewise provided by the invention. According to the coating method, at least one monolayer of a self-assembling protein is produced on a substrate by first providing a stabilized aqueous solution which comprises at least one self-assembling protein. To provide the coating solution, protein units aggregated from an aqueous solution of self-assembling proteins are separated off, and addition of a solution of ionic surfactants and/or of a salt-containing and/or alkaline and/or acidic solution to the protein-containing coating solution generates monomers or oligomers of the self-assembling proteins, and stabilizes them, the amount of ionic surfactant added being such that only the surface-active part of each active protein monomer or predominant protein oligomer is enveloped by surfactant particles. A substrate surface is then brought into contact with the stabilized, protein-containing solution, thus producing a protein-containing coating on the substrate. The supernatant solution is removed from the coated substrate and/or the coated substrate is dried.

Claims

1. Method for coating a substrate with a monolayer or a bilayer of self-assembling proteins, selected from hydrophobins or surface layer proteins or recombinant fusion proteins with at least two domains, wherein at least one domain is a hydrophobin or a surface layer protein and one domain is a functional domain, the method comprising the steps: i. providing a protein-containing coating solution, ii. contacting the surface of the substrate with the protein-containing coating solution, iii. removing a supernatant solution from the coated substrate and/or drying the coated substrate, characterized in that the step of providing the protein-containing coating solution includes: a) separating aggregated protein units from an aqueous solution of self-assembling proteins so that a concentration of the self-assembling proteins in the aqueous solution after separation of the aggregated protein units amounts to at least 5 ng/l, wherein the aqueous solution has a pH value of 7 to 9.5, and b) adding a solution of ionic surfactants in a range of 1 mmol/l to 10 mmol/l or in a range of 1 mol/l to 500 mol/l, the solution of ionic surfactants being added in portions, and c1) adding a salt-containing solution with bivalent metal ions or anions, wherein a concentration of the metal ions or anions in the aqueous solution amounts to 0.01 mmol/l to 10 mmol/l, or c2) adding bases or acids so that the pH value is shifted in a direction of the isoelectric point of the self-assembling proteins, to generate and stabilize monomers or oligomers of the self-assembling proteins in the protein-containing solution, wherein so much ionic surfactant is added that only the surface-active part of each active protein monomer or predominant protein oligomer is enveloped by surfactant particles.

2. Method according to claim 1, characterized in that the surfactants are selected from the group of hydrocarbon-coupled sulfates, sulfonates or carboxylates or from the group of quarternary ammonium compounds.

3. Method according to claim 1, characterized in that the salt-containing and/or alkaline and/or acidic solution contains alkaline earth metal ions or metal ions of the transition metal groups I and II in the form of chlorides, nitrates, carbonates, acetates, citrates, gluconates, hydroxides, lactates, sulfates, succinates or tartrates, or inorganic anions, in particular halides, hydroxides, nitrates, carbonates, sulfates, phosphates but also organic anions, in particular acetates, citrates, succinates or tartrates which have alkali metal ions as counter ions.

4. Protein-containing coating solution, containing monomers or oligomers of self-assembling proteins, selected from hydrophobins or surface layer proteins or recombinant fusion proteins with at least two domains, wherein at least one domain is hydrophobin or surface layer protein and one domain is a functional domain, wherein the surface-active part of the monomers or oligomers is enveloped by ionic surfactants, wherein the protein-containing coating solution is provided by: a) separating aggregated protein units from an aqueous solution of self-assembling proteins so that a concentration of the self-assembling proteins in the aqueous solution after separation of the aggregated protein units amounts to at least 5 ng/l, wherein the aqueous solution has a pH value of 7 to 9.5, and b) adding a solution of ionic surfactants in a range of 1 mmol/l to 10 mmol/l or in a range of 1 mol/l to 500 mol/l, the solution of ionic surfactants being added in portions, and c1) adding a salt-containing solution with bivalent metal ions or anions, wherein a concentration of the metal ions or anions in the aqueous solution amounts to 0.01 mmol/l to 10 mmol/l, or c2) adding bases or acids so that the pH value is shifted in a direction of the isoelectric point of the self-assembling proteins, to generate and stabilize monomers or oligomers of the self-assembling proteins in the protein-containing coating solution, wherein so much ionic surfactant is added that only the surface-active part of each active protein monomer or predominant protein oligomer is enveloped by surfactant particles.

5. Use of a protein-containing coating solution according to claim 4 for coating substrate surfaces.

6. Substrate comprising a protein-containing coating of at least one self-assembling protein, characterized in that on the substrate surface a monolayer or a bilayer of self-assembling proteins, selected from recombinant fusion proteins with at least two domains, wherein at least one domain is a hydrophobin or a surface layer protein and one domain is a functional domain, is arranged, on the surface of the monolayer or of the bilayer of self-assembling proteins a layer which contains at least one ionic surfactant is present, wherein the surfactant is present in coordinative bond with the self-assembling protein, wherein the functional domain is arranged on the side of the protein-containing coating that is facing away from the substrate.

7. Substrate according to claim 6, characterized in that the functional domain is a fluorescent protein domain, a catalytic domain, or a protein domain with enzyme activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) With the aid of the following figures and examples, the invention will be explained in more detail without limiting the invention thereto.

(2) FIG. 1 SEM image of a silicon wafer coated with a monolayer of the S-layer protein SbsC-(R5P).sub.2 from Bacillus stearothermophilus with deposited SDS layer. The homogeneous areas to the left and the right show the protein monolayer from which the SDS layer (shown in the middle) has been removed.

(3) FIG. 2 AFM image of a silicon wafer coated with a monolayer of the class 1 hydrophobin Ccg-2 from Aspergillus nidulans after removal of the SDS layer with exposed protein monolayer.

(4) FIG. 3 height profile of a silicon wafer coated with a monolayer of the class 1 hydrophobin Ccg-2 from Aspergillus nidulans determined by AFM.

DESCRIPTION OF PREFERRED EMBODIMENTS

Example 1: Expression of Recombinant Self-Assembling Fusion Proteins in E. coli and Purification

(5) The recombinant fusion proteins listed in Table 1 were generated which each have at the N terminal the self-assembling protein domain and at the C terminal a functional domain listed therein.

(6) TABLE-US-00001 TABLE 1 fusion protein self-assembling domain functional domain HFBI-(R5P).sub.2 class II hydrophobin subunit of silaffin HFBI (Trichoderma reesei) (Cylindrotheca fusiformis) HFBI- class II hydrophobin shortened subunit of silaffin (XSR5P).sub.3 HFBI (Trichoderma reesei) (Cylindrotheca fusiformis) Ccg2-HA class I hydrophobin human influenza Ccg2 (Neurospora crassa) hemaglutinin tag S13240-HA S-layer protein human influenza S13240 (Geobacillus hemaglutinin tag stearothermophilus) SbsC-HA S-layer protein human influenza SbsC (Geobacillus hemaglutinin tag stearothermophilus) SbsC-(R5P).sub.2 S-layer protein subunit of silaffin SbsC (Geobacillus (Cylindrotheca fusiformis) stearothermophilus) SbsC- S-layer protein shortened subunit of silaffin (XSR5P).sub.3 SbsC (Geobacillus (Cylindrotheca fusiformis) stearothermophilus) SslA S-layer protein no functional domain SslA (Sporosacina ureae)

(7) The DNA sequences coding for the respective domain are amplified by means of polymerase chain reaction (PCR) for this purpose and, by means of overlap PCR, a fusion gene was fused. The used flanking primers contained recognition sequences for the restriction endonucleases NdeI and XhoI. After vector and fragment restriction by means of the corresponding endonucleases, ligation, and transformation in Escherichia coli (E. coli) as a host organism, the presence of the fusion gene in the generated plasmids was checked.

(8) For the production of the fusion protein containing the self-assembling protein, the plasmids which contained the generated fusion gene correctly, were transformed in the E. coli strain SHuffle T7 Express. The fusion protein is concentrated in the bacterial cells intracellularly.

(9) The transformed E. coli cells were cultured at 30 C. The expression of the fusion gene was induced by addition of 0.4 mM (mmol/l) IPTG. After 4-6 h, the cells were pelleted by means of centrifugation (at 4,000g, 4 C., 10 min). After washing the cell pellet 2 times with 50 mM tris buffer (pH 7.5), the cells once more were pelleted by centrifugation. For cell disruption, the E. coli cells were subjected thrice to an ultrasonic treatment (9 cyc., 70%, 4 C., 2 min) and three times to a subsequent French Press treatment (20,000 psi). The disrupted bacterial cells were washed twice with 50 mM tris buffer (pH 7.5). For protein extraction the disrupted cells were taken up in 1 ml lysis buffer and incubated at 23 C. for 30 min. The purification of the fusion protein was carried out by means of nickel affinity chromatography. The elution of the fusion protein occurred in an isotype composed phosphate buffer (pH 4.5) with 250 mM imidazole.

(10) In order to transfer the protein into an active state, the obtained eluate was dialyzed against the 4.000-fold volume of a dialysis buffer at 4 C. for maximally 48 h. For the dialysis of hydrophobins a tris buffer (50 mM, pH 8.5 reduced with 1 mM glutathione and oxidized with 0.2 mM glutathione) was used. For the dialysis of surface layer proteins, 10 mM of tris buffer (pH 9.0) was used as a dialysis buffer.

Example 2: Production of a Protein Monolayer on a Silicon Wafer

(11) An ordered protein monolayer of a self-assembling protein, as obtained in Example 1, was generated on a silicon wafer. For this purpose, the fusion protein HFBI-(XSR5P).sub.3 was employed which contains a class I hydrophobin (HFBI from Trichoderma reesei) as a self-assembling domain and contains as a functional domain a human influenza hemaglutinin tag.

(12) First, for sedimentation of larger protein aggregates, a centrifugation (15 min at 18,000g, 4 C.) was carried out and the protein concentration determined (according to Lowry). The sediment was taken up in 50 mM tris buffer pH 8.5. A protein concentration of 100 ng/l was adjusted.

(13) To this protein-containing solution a filtered 8 mM sodium dodecylsulfate (SDS) was added in four single portions for a period of time of 10 min. For HFBI-(XSR5P).sub.3 the added surfactant quantity per liter of a solution adjusted to a protein concentration of 100 ng/l was 2 mmol so that a final surfactant concentration of 2 mmol/l was adjusted in the solution.

(14) Based on equation (3), for a 100 ng/l protein solution the surfactant concentration is to be adjusted to between 0.5 mmol/l and 5.6 mmol/l. The self-assembling fusion protein HFBI-(XSR5P).sub.3 has a molecular weight of about 14,000 g/mol. For a protein concentration in the solution of 100 ng/l, the concentration in mol/l accordingly amounts to 7.14*10.sup.6 mol/l. The surfactant head group of the used SDS molecule has an effective surface area of A.sub.TKG of 0.62 nm.sup.2. The protein molecule has a hydrodynamic radius R.sub.N of 2.7 nm. Based on this, a protein surface area of A.sub.O,protein=91.6 nm.sup.2 results from equation (4).

(15) The stabilized solution obtained in this manner was clear and was stable for at least five days when stored at 20 C.

(16) The stabilized solution which contains monomers of the self-assembling protein was used for coating a silicon wafer with edge lengths 11 cm. For this purpose, 100 l of the stabilized protein-containing solution was applied onto a silicon wafer with incubation for at least 30 min. Subsequently, the supernatant solution was drawn off and the silicon wafer coated with HFBI-(XSR5P).sub.3 washed in filtered, twice distilled water. The storage of the coated silicon wafer was done in the dialysis buffer mentioned in Example 1.

(17) Subsequently, the drawn-off stabilized protein solution was employed in other coating processes.

(18) In order to make accessible the functional domain of the self-assembling fusion protein on the substrate, remaining surfactant was removed from the coated substrate by means of precipitation reaction.

Example 3: Characterization of the Coating with HFBI-(XSR5P)3 on a Silicon Wafer Prepared in Example 2 by Means of Ellipsometry

(19) The coated silicon wafer obtained in Example 2 was examined by ellipsometry (Multiskop of the company OPTREL (Berlin)). The change of the polarization plane of a monochromatic laser beam (wavelength 632.8 nm) was determined by reflection at an angle of 136. By measuring the intensity of the perpendicular (R.sub.S) and parallel (R.sub.p) reflected planes of incidence of the polarized light beam, the intensity ratio () was determined. The ellipsometric angles and are derived from the FRENSEL equations. By including in the calculation the refractive indices of the single layers, the layer thickness of the uppermost layer was calculated.

(20) $=\frac{R_p}{R_s}=\frac{.Math.R_p.Math.}{.Math.R_s.Math.}{e}^{i\left({}_p-{}_s\right)}=\tan \left(\right)e^i$

(21) In this context, .sub.p and .sub.s indicate the phase difference to the zero point of light polarized perpendicular and in parallel. The optical constants known from the literature were used for silicon as a substrate and silicon dioxide. As refractive index of the protein film 1.375 was used.

(22) The determined layer thickness of HFBI-(XSR5P).sub.3 on the silicon wafer amounted to 13.20.2 . This corresponds to the literature values of a monolayer of the self-assembling protein.

(23) The following Table 2 shows the ellipsometric data of an analysis of four different specimens (1 to 4, the different values in the individual row were taken at different measuring points on the respective specimen):

(24) TABLE-US-00002 TABLE 2 1 2 3 4 .sub.SiO2 [] 175.87 175.772 175.761 175.879 d.sub.SiO2 [] 16.5 17.0 17.0 16.5 173.216 173.172 173.138 173.168 173.26 173.297 173.138 173.509 173.324 173.09 173.362 173.166 173.334 173.211 173.45 173.37 173.096 173.436 173.06 .sub.protein [] 173.301 173.154 173.213 173.346 standard 0.062 0.090 0.129 0.166 average deviation D.sub.protein [] 13.2 13.4 13.1 13 13.2 0.2

Example 4: Scanning Electron Microscopy (SEM) of a Silicon Wafer Coated with SbsC-(R5P)2

(25) A silicon wafer was coated analog to Example 2 with the self-assembling protein SbsC-(R5P).sub.2 (Table 1). The obtained coated silicon wafer was examined by means of SEM (Zeiss DSM 982 Gemini). For the analysis, the coated silicon wafer was dried and subsequently metallized with an atomic gold layer. The results of the SEM analysis are shown in FIG. 1. Bright areas indicated therein the protein layer with a deposited SDS layer. The dark areas indicate the protein as a monolayer from which the SDS layer has been removed.

Example 5: Atomic Force Microscopy (AFM) of a Silicon Wafer Coated with Ccg2-HA

(26) A silicon wafer coated analog to Example 2 with Ccg2-HA was examined in an aqueous environment by means of AFM. The results of the AFM analysis are shown in FIG. 2. A uniform protein monolayer can be seen. The Ccg2 tube structures have a length of about 100 nm (FIG. 2, bright areas). The height profile (FIG. 3) shows that the self-assembled structures of Ccg2-HA have a width of about 25 nm and a height of about 2.5 nm.

Example 6: Exposing Functional Domains for Self-Assembling Fusion Proteins

(27) With the self-assembling fusion protein HFBI-(R5P).sub.2 produced in Example 1, a silicon wafer was coated with a monolayer of the protein analog to Example 2. The layer thickness, which was determined ellipsometrically analog to Example 3, amounted to 13.80.4 . In the fusion protein the functional domain, the R5P subunit of silaffin, is fused with the hydrophilic part of the hydrophobin.

(28) It was checked with the aid of the coated silicon wafer by means of contact angle measurement whether the hydrophilic domain of the self-assembling protein and with it also the functional domain R5P is oriented toward the medium, i.e., on the side facing away from the silicon wafer. For this purpose, the contact angle in air was determined according to the sessile drop method (Drop Shape Analysis System DSA10, Krss GmbH, Germany). The contact angle in degree of a drop of 2 l of deionized water was determined.

(29) An uncoated silicon wafer cleaned with ethanol has a contact angle =35.22. The silicon wafer coated with HFBI-(R5P).sub.2 had a contact angle =56.40.7. This is clear evidence that the hydrophilic domain is exposed in the medium.

Example 7: Production of a Hydrophobin Bilayer on a Silicon Wafer

(30) An ordered protein bilayer of a hydrophobin, as obtained in Example 1, was generated on a silicon wafer. For this purpose, the fusion protein HFBI-(R5P).sub.2 was used which contains a class I hydrophobin (HFBI from Trichoderma reesei) as a self-assembling domain and as a functional domain a mineralization tag.

(31) First, for sedimentation of larger protein aggregates, a centrifugation (15 min at 18,000g, 4 C.) was performed and the protein concentration was determined (according to Lowry). The sediment was discharged. A protein concentration of 100 ng/l was adjusted.

(32) To this protein-containing solution, a filtered 10 mM sodium sulfate solution (Na.sub.2SO.sub.4) was added in four portions during a time period of 10 min. Subsequently, an 8 mM solution of sodium dodecylsulfate (SDS) is added. For HFBI-(R5P).sub.2 the added salt concentration was 0.05 mM and the surfactant quantity per liter of the solution adjusted to a protein concentration of 100 ng/l was 20 mol so that a final surfactant concentration of 20 mol/l was adjusted in the solution.

(33) The stabilized solution obtained in this manner was clear.

(34) The stabilized solution that contains multimers of the hydrophobin was used for coating a silicon wafer with edge lengths 11 cm. For this purpose, 200 l of the stabilized protein-containing solution was applied to the silicon wafer with incubation for at least 30 min. Subsequently, the supernatant solution was drawn off and the silicon wafer coated with HFBI-(R5P).sub.2 washed in filtered, twice distilled water. The storage of the coated silicon wafer was done in the dialysis buffer mentioned in Example 1.

(35) Subsequently, the drawn-off stabilized protein solution was used for further coating processes.

(36) In order to accessible the functional domain of the self-assembling fusion protein on the substrate

Example 8: Characterization of the Coating with HFBI-(R5P)2 and HFBI-(XSR5P)3 on a Silicon Wafer Produced in Example 6 by Means of Ellipsometry

(37) The coated silicon wafer obtained in Example 2 was examined ellipsometrically (Multiskop of the company OPTREL (Berlin)). The change of the polarization plane of a monochromatic laser beam (wavelength 632.8 nm) was determined by reflection at an angle of 136. By measuring the intensity of the perpendicular (R.sub.S) and parallel (R.sub.p) reflected planes of incidence of the polarized light beam, the intensity ratio () was determined. The ellipsometric angles and are derived by the FRENSEL equations. By including in the calculation the refractive indices of the single layers, the layer thickness of the uppermost layer was calculated.

(38) $=\frac{R_p}{R_s}=\frac{.Math.R_p.Math.}{.Math.R_s.Math.}{e}^{i\left({}_p-{}_s\right)}=\tan \left(\right)e^i$

(39) In this context, .sub.p and .sub.s indicate the phase difference to the zero point of light polarized perpendicular and in parallel. The optical constants known from the literature were used for silicon as a substrate and silicon dioxide. As an index of refraction of the protein film 1.375 was used.

(40) The determined layer thickness of HFBI-(R5P).sub.2 on the silicon wafer amounted to 25.71.8 , respectively 25.80.8 for the construct HFBI-(XSR5P).sub.3. These layer thicknesses correspond to the literature values of a bilayer of the hydrophobin.

(41) The following Table 3 shows the ellipsometric data of an analysis of seven different specimens (1 to Z), the different values in the individual rows were taken at different measuring points on the respective specimen):

(42) TABLE-US-00003 TABLE 3 Ellipsometric measured data for substrates coated with HFBI-(R5P).sub.2 1 2 3 4 5 6 Z .sub.SiO2 [] 175.975 176.063 175.282 175.894 175.858 175.923 175.47 d.sub.SiO2 [nm] 16 15.6 19.3 16.4 18.8 16.3 18.4 170.937 171.241 170.099 170.964 170.101 171.524 169.585 170.617 171.219 170.429 171.171 169.925 171.368 170.165 170.648 171.881 170.093 171.03 169.968 171.227 169.759 171.278 171.624 170.37 170.217 171.648 170.248 171.054 171.763 170.192 171.073 170.482 171.472 171.636 .sub.protein [] 170.907 171.533 170.237 171.055 169.998 171.413 170.048 standard 0.279 0.272 0.155 0.106 0.092 0.232 0.367 average deviation D.sub.protein [nm] 26.2 23.4 26 25 27.8 23.2 28 25.7 1.8

Example 9: Determination of the Exposition of Functional Domains for Self-Assembling Fusion Proteins

(43) With the self-assembling fusion protein HFBI-(R5P).sub.2 produced in Example 1, a silicon wafer was coated with a bilayer of the protein analog to Example 7. The layer thickness which was determined ellipsometrically analog to Example 8 amounted to 25.71.8 . In the fusion protein the functional domain, the R5P subunit of the silaffin, is fused with the hydrophilic part of the hydrophobin.

(44) It was checked with the aid of the coated silicon wafer by means of contact angle measurement whether the hydrophilic domain of the self-assembling protein and thus also the functional domain R5P is oriented toward the medium, i.e., on the side facing away from the silicon wafer. For this purpose, the contact angle in air was determined according to the sessile drop method (Drop Shape Analysis System DSA10, Krss GmbH, Germany). The contact angle in degree of a drop of 2 l of deionized water was determined.

(45) An uncoated silicon wafer cleaned with ethanol had a contact angle =35.22. The silicon wafer coated with HFBI-(R5P).sub.2 had a contact angle =54.10.9. This is clear evidence that the hydrophilic domain is exposed in the medium.

(46) to make, remaining surfactant was removed from the coated substrate by means of precipitation reaction.

Example 10: Examination of the Accessibility of the Fused HA Tag of a Silicon Wafer Coated with Ccg2-HA

(47) For determining the orientation of the deposited protein monomers on a silicon wafer coated with Ccg2-HA in analogy to Example 2, coupling of a fluorescence-marked antibody was carried out at the fused protein domain of a hydrophobin-fusion protein. For the fluorescence-microscopic analysis of the accessibility of the HA tag, the antibody anti-HA, AlexaFluor 488 conjugate (INVITROGEN GMBH; Darmstadt, Germany) was diluted in accordance with manufacturer's instructions and incubated for 30 min on the substrate. Subsequently, the substrate was washed three times with twice distilled water in order to remove residues of unbonded antibody. Fluorescence-microscopic images were performed with a Zeiss Axio Observer.Z1 (CARL ZEISS MICROIMAGING GMBH; Jena, Germany) in combination with the corresponding filter set 38 GFP. A green fluorescing substrate surface confirms the accessibility of the fused HA tag.

(48) As a reference, a substrate according to Example 2 was treated analogously with BSA. After incubation with the antibody anti-HA, AlexaFluor 488 conjugate, no green fluorescing signals were detectable on the substrate surface.

Example 11: Examination of the Accessibility of a Fused Luciferase Tag of a Plastic Surface Coated with HFBI-GLuc

(49) For determining the accessibility of protein domains of a fusion protein, coating of a silicon wafer with HFBI-GLuc was carried out in analogy to Example 2. The fused luciferase domain (GLuc) catalyzes the chemical reaction of luciferin with emission of light at a wavelength of 475 nm. In addition to a visual evaluation, the quantitative fluorescence analysis was carried out by means of Infinite M200 plate reader (TECAN GROUP LTD.; Mnnedorf, Germany). The unfused protein domains (HFBI or GLuc) serve as references.

(50) After the addition of luciferol, the bifunctional hydrophin chimera (HFB1-Gluc) as well as the subunit, as expected, showed a luminescence signal. The catalytically inactive HFB1 subunit does not show a luminescence signal. In a second experiment, the proteins were deposited on a polystyrene surface as in Example 2. Reaction buffer was overlaid onto the substrates and the reaction was initiated by the addition of luciferol. Exclusively the immobilized bifunctional hydrophobin chimera showed a significant luminescence signal.

Example 12: Examination of the Long Term Stability of a Protein-Containing Coating Solution Containing Ccg2-tRFP or HFBI-tRFP

(51) In order to examine the effect of ionic surfactant on the surface activity of the self-assembling proteins, CTAB was added to fluorescing fusion proteins. The fluorescing fusion proteins consist of a hydrophobin (Ccg2 or HFBI) which constitutes the self-assembling domain and a red fluorescing peptide domain (tRFP). The red fluorescing peptide domain enables examination of the effect of ionic surfactants on the structure of the fusion protein by means of fluorescence measurement. A change in the fluorescence intensity permits direct conclusions on conformation changes of the protein scaffolding which are caused by interactions with the ionic surfactant.

(52) Providing a protein-containing solution, containing a fusion protein (Ccg2-tRFP or HFBI-tRFP) or an unfused tRFP peptide sequence with the concentration 200 ng/L, was added optionally in four portions a solution of ionic surfactants, containing CTAB, to a final concentration of ionic surfactant of 1 mM. Subsequently, all solutions were filtered through a filter (0.22 m). The effect of the filtration, as well as the effect of added ionic surfactants was examined in the following for the time period of one week.

(53) Due to the properties of self-assembling proteins, the filtration of the protein-containing solutions, containing exclusively the fusion protein (Ccg2-tRFP or HFBI-tRFP) without addition of ionic surfactant, led to a reduction of the fluorescence intensity by 37%0 or 46%. However, the protein-containing solution, containing exclusively unfused tRFP without addition of ionic surfactant, showed no drop in the fluorescence intensity. The fluorescence intensity was controlled in the following every 24 hours for a time period of one week. All samples showed a continuous drop in the fluorescence intensity. After one week, in all samples a fluorescence was detectable that was reduced by approx. 95% relative to the initial value.

(54) The opposite result was obtained in case after addition of an ionic surfactant. The fluorescence intensity of the protein-containing solution, containing unfused tRFP peptide sequence and CTAB, dropped by 25%. The filtration of this sample had no effect on the fluorescence intensity. In case of the protein-containing solutions containing a fusion protein (Ccg2-tRFP or HFBI-tRFP), the addition of an ionic surfactant had no influence on the fluorescence intensity. Even after filtration of these solutions the fluorescence intensity remained unaffected. The fluorescence intensity was controlled in the following every 24 hours for a time period of one week. The protein-containing solutions, containing a fusion protein (Ccg2-tRFP or HFBI-tRFP) and an ionic surfactant, showed no drop in the fluorescence intensity across the entire testing period. The fluorescence intensity of the protein-containing solutions, containing keeping the unfused tRFP peptide sequence and an ionic surfactant, continuously dropped across the entire testing period. After one week, fluorescence was detectable in this sample that was reduced by about 67% relative to the initial value.