PROCESS FOR SEPARATING AND ANALYZING HYDROPHOBIC PROTEINS USING THIN LAYER CHROMATOGRAPHY

Abstract: A process for the separation and determination of strongly hydrophobic proteins, protein fragments and modifications, and of strongly hydrophobic peptides is described, which comprises dissolving the samples to be investigated in an organic medium, subjecting them to thin-layer chromatography and detecting hydrophobic proteins.

Description: PROCESS FOR SEPARAΗNG AND ANALYZING HYDROPHOBIC PROTEINS USING THIN LAYER CHROMATOGRAPHY Technical field The invention relates to a process for the detection and determination of strongly hydrophobic proteins, protein fragments and modifications, and of strongly hydrophobic peptides. Prior art The quantification of hydrophobic proteins is not possible or is only inadequately possible using the conventionally customary techniques. The methods customary for the separation of hydrophilic proteins, such as, for example, Western blotting are often only applicable to hydrophobic proteins to a limited extent, because on the one hand the separation on the SDS gel is inadequate and on the other hand the transfer of the proteins to the customary membranes can be described as at most semiquantitative. A separation of slightly modified proteins (e.g. methyl esters) cannot be effected by means of gel electrophoresis. The use of immunological methods, such as, for example, ELISA is very problematical, because as a rule this can only be carried out in aqueous systems. Organic solvents may react with the material of the microliter plates and render them unusable. The analytes to be quantified are usually present only in traces. Often, straight hydrophobic proteins are additionally associated with other lipophilic substances (e.g. lipids), which makes a quantification according to conventional methods impossible. In ELISA, the components are not separated from one another. Description of the invention The aim of the invention is the provision of a process which allows hydrophobic proteins, protein fragments and modifications and also strongly hydrophobic peptides to be separated and to be quantified. A further aim is to make available a process which makes possible the determination of, for example, proteins which are slightly modified by acetylation or oxidation. It is a further aim to indicate such a process which is suitable, in particular, for the determination of hydrophobic host-cell protein impurities (HCP) in the biotechnological production of hydrophobic proteins, such as, for example, the extremely hydrophobic lung surfactant protein SP-C. It has now been found that these aims are achieved by thin-layer chromatographic separation of the protein mixture dissolved in an organic medium and immunological detection of the separated proteins. The object of the invention is therefore a process for the separation and quantification of strongly hydrophobic proteins, protein fragments and modifications and also of strongly hydrophobic peptides, which comprises dissolving the samples to be investigated in an organic medium, subjecting them to thin-layer chromatography and rendering the hydrophobic proteins visible immunologically. Further objects are clear from the subclaims. It has surprisingly been found that for the thin-layer chromatographic separation step, procedures and materials known per se which were adapted to the hydrophobic properties of the proteins to be separated lead to the aim. Possible chromatography plates are all plates whose coating is suitable for the separation of hydrophobic mixtures in organic media. Of these, those which prove particularly suitable are the HPTLC plates marketed by Merck Darmstadt under the trade name Diol, which have a modified silica matrix. For hydrophobic proteins, suitable mobile phases for thin-layer chromatography are organic solvents and solvent mixtures, e.g. of chloroform and methanol. Mixtures of nonpolar and polar solvents are particularly expedient, possible nonpolar solvents, in particular, being chloroform, methylene chloride and toluene and polar solvents being short-chain alcohols, in particular methanol, ethanol and isopropanol. The application of the samples to the plates and the separation procedure are carried out in a customary manner, for example by means of commercially available automatic equipment. For preparation for immunological detection, the plates are dried after thin-layer chromatographic separation. For the saturation of nonspecific binding sites, the plates are incubated with a suitable blocking solution, e.g. gelatin or proteins. The plates are then incubated with the primary antibody. If this does not carry a label, detection can be carried out with the aid of a labeled second antibody. For detection, all commercially available detection processes can be used. After removal of excess first antibody by washing, incubation is carried out with a labeled second antibody. After washing, the labeled antibodies are detected. They are visualized in a customary manner, e.g. by addition of luminol and hydrogen peroxide, for example with the aid of the ECL (Enhanced Chemiluminescence) detection process of Amersham Buchler, which is very sensitive. In the purity analysis, with a view to slight chemical modifications, the substances can be detected on the TLC plates directly after separation also using customary protein staining reagents. In the following text, the invention is described by example with the aid of a process for the determination of the host-cell protein impurities in a biotechnological process for the preparation of r-SP-C by means of E. coli. Example 1. Obtainment of HCP for immunization purposes In agreement with procedures known from the literature and with relevant official regulations, an antigen fraction from the final phase of the downstream process was sought. Therefore, for the immunization, HCPs were withdrawn from a fermentation phase in which the r-SP-C is pure to 80 to 90%. For the removal of the HCP antigen fraction, 60 g of inclusion bodies isolated by filtration an/or centrifugation from a 10 1 blank fermentation were used after lyophilisation for about 96 hours. 2. Preparation of antisera 1 mg of HCP, dissolved in 95% strength isopropanol of pH 2, was dried in a vacuum concentrator (speedvac®), resuspended in 0.5 ml of phosphate-buffered saline, mixed with 0.5 ml of adjuvant (ABM-S for the base immunization and ABM-N for the booster injections) and in each case injected subcutaneously in an amount of 1 mg/rabbit. The immunization scheme was carried out according to standard protocols: after the primary immunization, booster injections were carried out every 4 weeks up to six times. Taking of blood was in each case carried out 10 days after the last injection in order to monitor the development of the titer. As soon as the titer was satisfactory, 50 ml of blood were taken and serum was prepared according to standard processes. 3. Determination of the titer For determination of the titer, the individual sera at various dilutions were analyzed by means of the new method described as immuno-thin-layer chromatography (immuno-TLC). Dilutions of 1:5,000, 1:10,000, 1:20,000 and 1:50,000 were used in order to analyze 4, 16, 62.5 and 250 ng of HCP. At a dilution of 1 :10,000, all rabbit antibodies investigated recognized HCP components in proportion to the amount of total protein. Antisera having a similar titer were pooled and reanalyzed. The serum was characterized according to standard processes and stored in aliquots at -20°C. 4. Sample preparation and thin-layer chromatography (TLC) The samples to be analyzed were dried in a vacuum concentrator and dissolved in 20 to 200 μl of CHCb/MeOH. For thin-layer chromatography, HPTLC plates having a modified silica matrix, such as are marketed by Merck Darmstadt under the trade name Diol, were used. The application of the samples to the HPTLC plates was carried out automatically using a Lingomat IV (Camag, Berlin). After sample application, the plates were air-dried and subjected to chromatography using a CHCiyMeOH mixture [CHCIa/MeOH/25% strength NH 4 OH/H 2 0 = 32.5/15/1/2 (ratio by volume)] as the liquid phase. After chromatography, the plates were dried. 5. Immunostaining of HCP with anti-HCP antibodies For the saturation of nonspecific binding sites, the dried HPTLC plates were incubated for 4 hours with 3% strength fish gelatin in PBS which contained 150 mM NaCl, 12 mM Na 2 HPO< and 3 mM NaH 2 P04 (pH 7.4). The plates were then incubated overnight and lightly shaken in the presence of the primary antibody, usually at a dilution of 1 :10,000. Unbound antibodies were removed by washing several times with TBS/T from 4 mM tris HCl, 100 mM NaCl, 0.05% Tween 20 (pH 7.4). For hybridization with the primary antibody, the plates were incubated for 2 hours with horseradish peroxidase-conjugated secondary antibody at a dilution of 1 :80,000 in the TBS/T. Unbound antibodies were removed as described above by washing the plates several times with TBS/T. Immunoreactive complexes were visualized using an ECL detection system from Amersham Buchler. The plates were incubated for 20 to 60 seconds with an X-ray film (Hyperfilm Amersham). 6. Staining and immunostaining of SP-C In order to detect modifications of SP-C (e.g. methyl ester at the C-terminus and methionine sulfoxide), the protein forms separated by means of TLC can be detected by means of staining with Ponceau S. A more sensitive possibility is the use of SP-C antibodies and of the method indicated above in analogy to HCP determination. 7. Video-imaging of the X-ray films and computer analysis For the quantification of the immune complexes, the X-ray films were digitalized using a video-imager (Cybertech, Berlin, Germany). The signal intensities on the X-ray films were analyzed by computer analysis using Wincam software (Cybertech). 8. Quantification of HCP For the determination of HCP in r-SP-C samples, aliquots were analyzed pure or after addition of 2.5, 5.0 or 10.0 ng of HCP. The amounts of HCP in the individual samples were determined by linear regression calculations. Percentages were determined by means of the formula % = (ng of HCP x 100)/ng of r-SP-C. In order to quantify small amounts of HCP in r-SP-C, it is necessary to separate the majority of the target protein from the small amount of HCP. This is effected by thin-layer chromatography. The detection limit for HCP was analyzed by mixing 5 μg of highly pure r-SP-C with ng amounts of HCP. Under these conditions, 0.125 ng of HCP was the lowest amount which it was possible to differentiate from the endogenous HCP content of r-SP-C. The amount of the sample which can be analyzed by immuno-TLC depends on the HCP content. For r-SP-C samples which contain less than 0.1% of HCP, 1 to 5 μg of r-SP-C are analyzed. Since it is possible to separate amounts of r-SP-C up to 20 μg by thin-layer chromatography and the quantification limit is 1 ng on luminography (when using 5 μg of added r-SP-C), it was possible to detect HCP theoretically down to 0.005%. The detection of 0.002% HCP appears to be a reliable quantification limit under standard conditions.

Claims: Patent claims 1. A process for the separation and determination of strongly hydrophobic proteins, protein fragments and modifications, and of strongly hydrophobic peptides, which comprises dissolving the samples to be investigated in an organic medium, subjecting them to thin-layer chromatography and detecting the hydrophobic proteins. 2. The process as claimed in patent claim 1 , wherein the detection is carried out by means of immunological methods. 3. The process as claimed in patent claim 1 , wherein the detection is carried out by means of staining methods. 4. The process as claimed in patent claim 1 , wherein coated plates are used for thin-layer chromatography using a modified silica matrix. 5. The process as claimed in patent claim 1 , wherein the organic medium used is a mixture of solvents in which strongly hydrophobic proteins or peptides are soluble. 6. The process as claimed in patent claim 5, wherein the organic medium used is a mixture of chloroform and methanol.

Inventors: ISE WOLFGANG, NAVE RUEDIGER, STEINHILBER WOLFRAM, ISE, WOLFGANG, NAVE, RUEDIGER, STEINHILBER, WOLFRAM
Applicants: BYK GULDEN LOMBERG CHEM FAB, ISE WOLFGANG, NAVE RUEDIGER, STEINHILBER WOLFRAM, BYK GULDEN LOMBERG CHEMISCHE FABRIK GMBH, ISE, WOLFGANG, NAVE, RUEDIGER, STEINHILBER, WOLFRAM
Application year: 1997
Main classification: C07K14
IPC codes: C07K14, G01N30, G01N33