Method for lysing cells and tissue by employing a polymer and the use of the polymer for this method

Abstract

Described is a method for lysing cells and tissue by employing certain polymers as well as the use of these polymers for totally or partially lysing cells and tissue.

Description

[0001] METHOD FOR LYSING CELLS AND TISSUE BY EMPLOYING A POLYMER AND THE USE OF THE POLYMER FOR THIS METHOD

[0002] Description of the invention

[0003] The present invention relates to a method for lysing cells and / or tissue by employing a polymer and the use of the polymer for this method.

[0004] Background of the invention

[0005] Cell lysis is an inevitable step for analytical approaches of biomolecules, such as soluble and membrane proteins, nucleic acid and other molecules in all kind of downstream applications. To archive reproducible and best possible results, a native conformation of the extracted proteins is a huge advantage. Membrane associated interaction partners, protein complexes and receptorligand couples benefit of the natural folding present in the cell membrane. State of the art approaches build on lysis buffers containing NP-40, SDS and deoxycholate often in combination with mechanical procedures to ensure an enhanced cell lysis causing non-native conditions without the essential lipid environment, as well as mechanical methods, like a french press and sonication.

[0006] Especially for the preparation of DNA the alkaline lysis has been established since about forty years. In this procedure an alkaline solution and SDS are applied with moderate mechanical stress in order to purify plasmid DNA without genomic DNA impurities.

[0007] For protein extraction from mammalian cells RIPA buffer, a mixture from SDS, sodium chloride, sodium deoxycholate, octylphenoxypolyethoxyethanol and tris buffer, is established. But this procedure contains many drawbacks, as proteins become denatured by SDS and sodium deoxycholate. Therefore, this method cannot be used for applications where native proteins with intact activity are required.

[0008] Mild detergents like DDM can be applied to lyse mammalian cells, but even under these conditions native proteins cannot be prepared.

[0009] With digitonin mammalian proteins can be solubilized and the protein can be obtained in the native state, but digitonin has limitations in solubility and so the solubilization procedure is still tedious. Digitonin is a natural product with variations from one charge to the next, but the most important disadvantage is, that additional lysis, whether mechanical or chemical, is required. Description of the invention

[0010] The technical problem underlying the present invention is to provide a method for lysing cells and tissue, which can be carried out quickly and easy, which is highly effective and which lysis the cells and tissues reliably.

[0011] This has been achieved by the subject-matter as defined in the independent claims. Preferred embodiments are defined in the dependent claims.

[0012] According to the present invention, there is provided a method for lysing cells and tissue, wherein the cells and the tissue are brought into contact with a polymer.

[0013] The term "lysis” as used according to the present invention refers to the breakdown of a cell caused by damage to its plasma (outer) membrane. It is pointed out that the term "lysis” used according to the present invention does envisage that from a pool of cells at least a part of the cells of the pool of cells up to all cells of the pool of cells are lysed. According to the method of the present invention, it is caused by certain polymers as will be defined later.

[0014] The term "cells and / or tissue” as used according to the present invention means that by the use of the certain polymers, cells, tissue and a mixture of the cells with the tissue are lysed by the use of the polymers. The tissue is a biological organizational level between cells and a complete organ. A tissue is an ensemble of similar cells and their extracellular matrix from the same origin that together carry out a specific function,

[0015] According to the method of the present invention, the cells and / or the tissue are brought into contact with a certain polymer. This step can be carried out in a medium, for example an aqueous medium, like salt solutions or buffer solutions. The detailed composition of the medium is chosen so that neither the cells or the tissue nor the polymer are negatively affected to carry out the lysis.

[0016] In one embodiment, the cell is an eucaryotic cell. It has been surprisingly found that the lysis of bacterial cells (Gram positive / - negative) as well as yeast was not observable.

[0017] The polymer can be a homo polymer or a copolymer. In one embodiment, the polymer can have hydrophilic groups, such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholines, and hydrophobic groups, such as polymerized styrene groups, polymerized diisobutylene groups, or linear Cl to C16 (like methyl and ethyl) aliphatic groups, branched Cl to C16 (like isopropyl or t-butyl) aliphatic groups and cyclic C5 to C12 aliphatic or aromatic groups.

[0018] In one embodiment, the polymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene maleic acid copolymer, (acrylic acid -co-styrene) copolymer and amphipol, which are in particular suitable to lyse the cells or the tissue.

[0019] The molecular weight of the polymer employed according to the method of the present invention can be 1900 to 20000, for example 2000 to 18000, or 2000 to 15000, or 4000 to 16000, or 4000 to 13000 or 5000 to 14000. The molecular weight can be measured gel permeation chromatography and mass spectrometry.

[0020] Examples for the polymers can be, but are not limited to styrene / maleic acid copolymers, sold by the trade name „SMA", derivatives of styrene / maleic acid copolymers like SMA 200 and 300, styrene / maleimide copolymers, like SMA 502. These substances can also be functionalized on the COOH groups, with amines, like ethanol amine or ethylene diamine to amides, or with alcohols like glycerol to esters. The polymers can also be functionalized with polyethylene glycols to esters and with aminated polyethylene glycols to amides.

[0021] The polymer can be diisobutylidene / maleic acid copolymers, for example DIBMA 10 and DIBMA 12 from Cube Biotech, derivatives of diisobutylidene / maleic acid copolymers, like DIBMA Gly, DIBMA Glu, Glyco DIBMA, and diisobutylidene / maleimide copolymers. DIBMA copolymers can be functionalized with the same molecules like SMA.

[0022] Further polymers can be copolymers from styrene and acrylic acid, in particular with a molecular weight of 5.500 and 11.000 and a relation acrylic acid / styrene of 45% / 55% to 55% / 45%, sold under the name „AASTY".

[0023] Modified polymers from polyacrylic acid can be used, where 10-90% of the carboxylic acid groups can be modified to amides with cyclooctylamine, 2-cyclohexyl-ethylamine, and the like. These substances are sold under the name „Amphipol Ultrasolve"

[0024] In addition to the above disclosure of the polymer, in the following a further description of the polymer is given. Examples for hydrophilic groups could be, but are not limited, to polymers of acrylic acid and methacrylic acid, maleic acid, carboxylic acid groups in general, amides with a, co alkylene diamine, co-hydroxyalkyl amine and co-aminoalkylthiols, trimethylammonio-alkylamin, amide from carboxylic acid groups with amino-glycerol TRIS, or Bis-Tris, amide with maltosamine, glucosamine, mannosamine and other amino-functionalized carbo hydrates, taurine.

[0025] Also, esters of carboxylic acid groups with polyethylene glycols, diols, triols, polyols, and carbo hydrates can be mentioned.

[0026] Other examples can be maleimides, with the nitrogen atom functionalized with alkyl chains with alcohol, thiol, amine, ammonium salts and the like.

[0027] Alternatively, zwitterionic molecules, consisting of ammonium and phosphate groups, ca be linked onto carboxylic groups, like it is described in US2020281855A1 or US2021171673A1.

[0028] Examples for hydrophobic groups could be, but are not limited, to polymerized styrene and derivatives, such as methylstyrene, diisobutylene and linear and branched alkanes, like 2-propyl, hexyl, octyl, or decyl, coupled to carboxylic groups via ester or amide functions. Also, maleimide groups with alkyl or aryl groups on the amino function are suited examples.

[0029] An example for the synthesis of styrene-maleic acid copolymers can be seen in Shintaro Sugai, Nobumichi Ohno, Conformational transitions of the hydrophobic polyacids, Biophysical Chemistiy, Volume 11, Issues 3-4, June 1980, Pages 387-395

[0030] The use of SMA for building a complex with lipids is described in WO 2006 / 129127 and references therein.

[0031] SMA can be purchased at Orbiscope or Cube Biotech, as SMALP 140, SMALP 200, or SMALP 300. DIBMA:

[0032] The synthesis of copolymers from diisobutylene and maleic acid anhydride is described in US 4,250,289 by BASF. Hydrolysis of anhydride copolymer to diisobutylene-co- maleic acid is described in Lee, Nature Protocols Vol. 11, No. 7, 2016, 1149-1162, which is described for SMA copolymer, but can be applied to DIBMA without problem. The synthesis of a DIBMA polymer with a functionalization of a glucosamine on 50% of all carboxy groups can be found on: B. Danielczak , M. Rasche, J. Lenz, E. Perez Patallo, S. Weyrauch, F. Mahler, M. Tope Agbadaola, A. Meister, J. Oyebamiji Babalola, C. Vargas, C. Kolar and S. Keller, A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscsf, DOI: 10.1039 / D1NR03811G (Paper) Nanoscale, 2022, 14, 1855-1867.

[0033] DIBMA can be purchased at Cube Biotech as DIBMA 10 and DIBMA 12.

[0034] The preparation of poly(aciylic acid-co-styrene) copolymers is described in WO 2020 257637 and S. Harrisson, F. Ercole and B. W. Muir, Living spontaneous gradient copolymers of acrylic acid and styrene: one-pot synthesis ofpH-responsive amphiphiles, Polym. Chem., 2010, 1, 326-332

[0035] Sometimes the copolymer is a copolymer from styrene and acrylic acid, or a copolymer from styrene and an acrylic acid derivative. Any copolymer derivative may find use in the subject copolymers. Examples for derivatives are aciylates, methacrylates, acrylic esters, acrylamides, and N-substituted acrylamides. In certain cases, the acrylic esters or acrylamides are substituted with a zwitterionic species, as described in US patent application 20190062469A1, the disclosure of which is incorporated herein by reference.

[0036] In certain embodiments the copolymer contains acrylic acid or an acrylic acid derivative content of from 30% to 70%, 35 to 65%, or 40 to 60%.

[0037] The synthesis of Amphipol is described in WO 115083 and in Marconnet, A, Michon, B., Le Bon, C., Giusti, F., Tribet, C., & Zoonens, M. (2020). Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules. doi:10.1021 / acs.biomac.0c00929.

[0038] Additional polyacrylates, modified with alkyl groups like pentyl, hexyl, and tert-butyl, are described in US 2020 / 0383918.

[0039] Polymethacrylate, containing butyl Methacrylate (BMA) in Copolymer: ~0.52 and methyl acryloxy choline (MAC) in Copolymer: ~0.48, with a degree of polymerization (DP): ~39.00, is distributed by Avanti Polar Lipids, with the brand name Polymethacrylate Copolymer (N-C4-52-6.9). Other polymethacrylates are described in Yasuhara K, Arakida J, Ravula T, Ramadugu SK, Sahoo B, Kikuchi JI, Ramamoorthy A. 2017. Spontaneous Lipid Nanodisc Formation by Amphiphilic Polymethacrylate Copolymers. J Am Chem Soc. 139(51):18657-18663. Polyacrylate polymers, modified with alkanes, such as n-butyl, t-butyl, pentyl, neopentyl, and hexyl are described in N. Z. Hardin, T. Ravula, G. Di Mauro, A. Ramamoorthy, Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodisks, Small. 2019 March; 15(9): el804813. doi:10.1002 / smll.201804813, and US2020383918A1.

[0040] Alternatively, linear carbo hydrates with a polymerization degree of less than 100, functionalized with hydrophobic groups, are mentioned in US2022 093587A. Examples for linear carbo hydrates are inulin, and examples for hydrophobic groups are alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalkyl having 1-3 hetero atoms. The hydrophobic group is bound to the carbo hydrate via an ether, ester, or amide group.

[0041] In another embodiment the concentration of the polymer (v / v) is about 0,01 % to about 5 %, in particular about 0.1 % to about 2,5 %.

[0042] The solution of polymers like DIBMA, SMA, AASTY, Ultrasolute Amphipol are able to dissolve cells and tissues very quickly.

[0043] In a further embodiment, the cell and / or the tissue is brought into contact with the polymer during about 10 seconds to about 5 minutes. It has been found that even after such a short contact time, the cells and the tissue are sufficiently lysed.

[0044] As pointed out above, with the method according to the present invention, eucaryotic cells are lysed selectively, whereas no lysis of procaryotic cells and yeast has been observed. This surprising finding allows that in one embodiment the method according to the present invention can be employed for separating eucaryotic cells or cell components from procaryotic cells or cell components. This can be for example achieved in that the cells are lysed and then the intact procaryotic cells or yeast cells are separated by known methods from the lysed fragments of the eucaryotic cells.

[0045] The present invention further relates to the use of the polymers as defined above for lysing cells and tissue. For the details of this use, it is referred to the above description of the materials, like cells and polymers, as well as to the method steps in their entirety. The present invention, in particular in the above-described embodiments, have several advantages. First, it is provided an easy to use, highly effective, fast and reliable lysis method which additional can stabilize membrane proteins in their unique lipid environment. This can be achieved by utilising different concentration (v / v) of 0.1% to 5% of the polymers in the buffer employed for the lysis. The employed lysis buffer (the polymer present in a buffer) ensures a time efficient and stable solubilization and stabilization of target proteins to study protein-protein interactions, the activity of native membrane proteins and other functional studies. Besides membrane proteins, soluble proteins can also be prepared in their native state. With native proteins, interaction analyses, such as immunoprecipitation and mass spectrometry, binding to specific antibodies, analyses of enzymatic activity, control of metabolic processes, and other functional studies.

[0046] Proteomics is the study of the proteome, i.e. the totality of all proteins present in a cell or living organism under defined conditions and at a defined time. In contrast to the (rather) static genome, the proteome is (highly) dynamic and can therefore change in its qualitative and quantitative protein composition due to changing conditions (environmental factors, temperature, gene expression, drug administration, etc.). The polymers of the invention allow a variety of proteins to be obtained in the native state by efficient and mild lysis, making these compounds ideally suited for use in proteomics.

[0047] Higher copolymer concentration (1% - 5%) leads to an effective cell lysis and membranes solubilization in less than 10 minutes, resulting in the release of intact, long-chained DNA and RNA. No mechanical support, which can destroy the nucleic acid chains, is necessary in this process.

[0048] This is a strong advantage for "next generation sequencing” approaches, where long read lengths are desired. The long-chain DNA or RNA obtained by the process of the invention can be used to sequencing methods, such as nanopore sequencing, which is optimized for high read lengths. Alternative sequencing technologies for which the nucleic acid obtained here can be used include single molecule real time sequencing (SMRT), pyrosequencing, reversible dye terminator sequencing, and sequencing-by-ligation mediated by ligase enzymes.

[0049] In a further embodiment, lower copolymer concentrations (0,01 - 0,1%) lead to a mild but yet effective cell lysis without effecting the nucleus resulting in an easy to process sample in less than 5 min. In this case RNA fragments like mRNA, tRNA and rRNA can be purified and prepared without major genomic DNA impurities. So downstream applications with the RNA fractions, proteins and other biomolecules, such as metabolites, obtained from the cytosol, are faster and more efficient.

[0050] The present invention further relates to the preparation of DNA and RNA after lysis of cells. The nucleic acid, especially DNA, is not attacked and remains long-chained and intact, due to the mild lysis conditions. The DNA and RNA thus obtained have little mechanical shear due to mild lysis and therefore have fewer breaks and are more intact compared to conventional lysis methods.

[0051] The method according to this invention is not restricted to the preparation of nucleic acids. After lysis, also proteins and other biomolecules can be prepared. The mild lysis method according to this invention allows, for example, the recovery of a large number of native proteins for proteomics applications such as MALDI mass spectrometry, affinity proteomics, microarrays, and the like. The protein samples can be used e.g., for expression proteomics and drug discovery.

[0052] Further, it is possible to lyse so that the cell envelope is lysed and the nucleus remains intact This is an important advantage in nucleic acid purification. While eukaryotic cells are lysed, bacterial cells (E Coli; Gram-positive) remain intact This advantage allows easy separation of bacteria and eukaryotic cell components.

[0053] In a further embodiment, even lower copolymer concentrations (0,005 - 0,05%) lead to a mild perforation of cells, leaving the biggest part of the cells alive. The concentration of the polymers and the incubation time can be adjusted, so that more than 75% of the remain alive, preferably more than 90% of the cells remain alive, more preferably more than 95% of the cells remain alive, his can be used for many applications, e.g., for the uptake of active substances into the cells.

[0054] In cell biology, transfection is the introduction of foreign DNA or RNA into animal and sometimes other eukaryotic cells. Methods for transfection contain coating or stabilization of the nucleic acid in order to facilitate the uptake, with substances like cyclodextrins, liposomes, dendrimers, or cationic polymers. Physical methods are using some physical means to force the transfected material into the target cell's nucleus. This sometimes work with a disrupt of the cell membrane, e.g., with electroporation, or magnetofection.

[0055] So, in a further embodiment, the copolymers in low concentrations (0.005-0.05%) lead to a disruption of the cell membranes without a total lysis of the cells and cell death, allowing the nucleic acid in a naked or modified form to enter the cells. The polymers of the invention can also be used together with substances that support the entry of the nucleic acid into cells, such as lipids, cyclodextrins, dendrimers, or functionalized polymers.

[0056] In diagnostics, biological components such as DNA, RNA, proteins and metabolites are examined qualitatively and quantitatively. This provides information about diseases, genetic predispositions, or the state of health. Diagnostic tests can be performed by medical professionals, but also by private individuals. Due to their ability to mildly lyse cells and tissues, to preserve nucleic acids with a low level of fragmentation, and to preserve proteins in their native state, the polymers of the invention are particularly suitable for diagnostic tests.

[0057] In a further aspect of the invention, the polymers can also be used in the form of a kit to make it easier for customers to use these substances.

[0058] Other possible components of such a kit include:

[0059] -A reducing agent included in the reagent can be any reducing agent that is known to reduce disulphide bonds in cellular proteins. Examples of such reducing agents are mercaptans, and preferred mercaptans are DTT (dithiothreitol), beta-mercapto ethanol, and tris(2-carboxy- ethyljphosphine (TCEP). Dithiothreitol and 3-mercaptoethanol are particularly preferred. The concentration of reducing agent in the reagent in accordance with this invention is from about 1 mM to about 10 mM, preferably from about 1 mM to about 5 mM.

[0060] -A chelating agent can be included that will disrupt secondary and tertiary structures of RNA.

[0061] The preferred concentration will be from 0.2 mM to 5 mM, and more preferably about 1 to 2 mM. Preferred chelators can be EDTA (ethylene diamine tetraacetic acid), EGTA (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriamine-pentaacetic acid), citric acid, histidines, and 8-hydroxyquinol.

[0062] -Buffer components, such as HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), MES, 2-(N-morpholino)ethanesulfonic acid, Tris [tris(hydroxymethyl)aminomethane], or Bis-Tris 2- [Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-l,3-diol], stabilize the pH of the solution in the physiological range, or between 6.5 and 8.0, preferably between 7.5 and 8.0. The concentration of the buffer is 1 mM to 250 mM, preferably 25 mM to 100 mM.

[0063] -lithium- sodium, or potassium salts, or mixtures thereof, are preferred to maintain the ionic strength of the mixture. This will help stabilizing the proteins and nucleic acids and other biomolecules. The concentration of the alkaline halides should be 1 mM to 200 mM, preferred 10 to 100 mM, more preferably 20 to 50 mM. The invention will be further illustrated by the following examples and figures. It is emphasized that the examples and figures shall not be construed to limit the invention thereto.

[0064] Figure 1 shows the lysis of HEK cells after 1 min / 10 min treatment with either RIPA buffer, Ultrasolute Amphipol 0,1% / 1% / 2,5% or control buffer.

[0065] Figure 2 shows detectable dead HEK cells after 1 min / 10 min / 30 min treatment

[0066] Figure 3 shows SDS PAGE analysis of HEK cell supernatant after 1 min / 10 min / 30 min treatment.

[0067] Figure 4 shows SDS PAGE analysis of eukaryotic cell lysis with different copolymers after different lysis time.

[0068] Figure 5 is a diagram showing the eukaryotic cell lysis with AASTY 50, comparison RIPA-AASTY 6-50, protein quantity (RIPA: 100%)

[0069] Figure 6 is a diagram showing the detectable dead HEK293 cells after treatment of up to 30min with either RIPA buffer, Ultrasolute Amphipol, SMA, AASTY, DIBMA or control buffer. 100%: Amount of cells offered

[0070] Figure 7 shows SDS PAGE analysis of prokaryotic cell lysis with different copolymers, protein gel with no proteins detected

[0071] Figure 8 shows SDS PAGE analysis of heart tissue lysis with Ultrasolute Amphipol 18.

[0072] Figure 9 shows SDS PAGE analysis of eukaryotic cell lysis with different copolymers, protein gel with / without lysed cell core

[0073] Examples

[0074] Example 1:

[0075] A total number of countable HEK cells after 1 min / 10 min treatment with either RIPA buffer, Ultrasolute Amphipol 0,1% / 1% / 2,5% or control buffer. Lower cell count equals fully lysed cells. The less cells are countable the more sufficient the cells lysis takes place (no organelles detectable - release of intact, long-chained DNA). A 1 min incubation with 0,1% Ultrasolute Amphipol 18 results in an equivalent amount of fully lysed cell count compared to RIPA buffer. A longer incubation time of 10 min results in a 50% reduction of detectable cells and therefore higher count of fully lysed cells compared to a short 1 min incubation. Longer RIPA buffer incubation times though do not show a change in cell count Higher concentrated Amphipol Ultrasolute 18 solutions result in even higher fully lysed cell counts (4,4x higher at 1% / 15, 7x higher at 2,5% compared to RIPA buffer at 10 min)

[0076] Example 2:

[0077] Detectable dead HEK cells in percent after 1 min / 10 min / 30 min treatment with either RIPA buffer, Ultrasolute Amphipol 0,1% or control buffer. Already after 1 min of incubation 61% of the cells treated with 0,1% Ultrasolute Amphipol are found dead. After 10 min 69% and after 30 min 88% are found dead. RIPA buffer and buffer control show a 2-5% dead cell count

[0078] Example 3:

[0079] SDS PAGE analysis of HEK cell supernatant after 1 min / 10 min / 30 min treatment with either RIPA buffer, Ultrasolute Amphipol 0,1% or control buffer. After incubation intact cells and cell debris were centrifuged and equal volumes of supernatant were run via SDS PAGE and detected via Coomassie staining. Compared to the buffer control Ultrasolute Amphipol 0,1% treated cells can be successfully lysed after as short as 1 min in comparable amounts as cells treated with RIPA buffer. A longer incubation time of 10-30 min results in better cell lysis compared to RIPA buffer shown by the stronger corresponding bands in SDS PAGE. Ultrasolute Amphipol outperformes RIPA buffer already in low concentration (0,1%) as soon as 10 min incubation time.

[0080] Example 4: Eukaryotic Cell Lysis with different Copolymers

[0081] In order to determine the solubilization efficiency, a SDS PAGE analysis of HEK293 cell supernatant after 1 min - 30 min treatment with either RIPA buffer (Thermo Fisher, Waltham, MA, USA), Ultrasolute Amphipol 18, SMALP 200, AASTY 6-50, DIBMA 10 (all Cube Biotech, Monheim, Germany) or control buffer is performed. A lower cell count equals fully lysed cell. The less cells are countable the more sufficient the cells lysis takes place (no organelles detectable - release of intact, long-chained DNA). After incubation intact cells and cell debris were centrifuged and equal volumes of supernatant were run via SDS PAGE and detected via Coomassie staining (Fig. 4).

[0082] The quantification is done by SDS PAGE analysis of HEK293 / T.ni. cell supernatant after 10 min treatment with either RIPA buffer, Ultrasolute Amphipol, SMALP, AASTY, DIBMA or control buffer. After incubation intact cells and cell debris were centrifuged and equal volumes of supernatant were run via SDS PAGE and detected via Coomassie staining.

[0083] With Polymer (AASTY 6-50 or Ultrasolute Amphipol 18 a ~20% (HEK293) / ~50% (T.ni.) increased protein amount can be identified via SDS PAGE band analysis. As a standard RIPA has been set to 100% (Fig.5).

[0084] Figure 6 shows detectable dead HEK293 cells after treatment of up to 30min with either RIPA buffer, Ultrasolute Amphipol, SMA, AASTY, DIBMA or control buffer. 100%: Amount of cells offered

[0085] Example 5: Procaryotic Cell Lysis with different Copolymers

[0086] To test the solubilization efficiency, a SDS PAGE analysis ofE. coli / B. subtilisceW supernatant after 1 min - 16h treatment with either RIPA buffer, Ultrasolute Amphipol 18, SMALP200, AASTY 6-50, DIBMA 10 or control buffer is performed (Fig.7 J. After incubation intact cells and cell debris were centrifuged and equal volumes of supernatant were run via SDS PAGE and detected via Coomassie staining. In comparison to eucaryotic cells no distinct protein bands are detected, only smear. Treated cells are still able to form colonies when plated on agar plates.

[0087] - 200 pl bacterial suspension were taken from a flask with an OD600 of 1.

[0088] - the reaction tubes were centrifuged at 2500g, 5 min, RT

[0089] - the resulting clear supernatant was discarded

[0090] - the resulting pellets were resuspended in 200pl of buffer containing buffer / RIPA / 2,5% (w / v) polymer solution

[0091] - the reaction tubes were incubated on ice for different amounts of time

[0092] - a centrifugation step at 14000g, 15min, RT was performed

[0093] - 25 pl of the supernatant was added to SDS sample buffer and 20 pl of the sample was loaded to an SDS PAGE.

[0094] Example 6: Heart Tissue Lysis with Ultrasolute Amphipol 18

[0095] Blue Native Page analysis of bovine heart tissue treated with either Ultrasolute Amphipol 18 or a buffer control. Both samples have been incubated for lh at RT with either a 1% (w / v) Ultrasolute Amphipol solution of a buffer control followed by an ultracentrifugation step at 100k g for lh at 4°C. 5 pl of the resulting supernatant have been applied to a Blue Native Page. Ultrasolute Amphipol 18 treatment resulted in the solubilization of a broad range of different sized proteins / protein complexes while the buffer control only shows one distinct band (Fig. 7).

[0096] Example 7: Eukaryotic Cell Lysis with different Copolymers, leaving the cell core intact

[0097] In order to test the solubilization efficiency, a SDS PAGE analysis of HEK293 and Sf9 cell supernatant after 10 min treatment with 0.01% to 2.5%, of either RIPA buffer, Ultrasolute Amphipol 18, SMALP 200, AASTY 6-50, DIBMA 10 or control buffer is performed (Fig.9). After incubation intact cells and cell debris were centrifuged and equal volumes of supernatant were obtained. If 0.01% to 0.05% are used with ten minutes incubation time, there is no increase of viscosity. If 2.5% polymer concentration is used for ten minutes incubation time, the lysate turns into a highly viscous fluid. High viscosity is an indication of free DNA and therefore lysed cell cores. After digestion with Benzonase (cat no.71206, Merck Millipore, 4°C for 30 min, used as described in producer protocol) the viscosity is reduced, so that the solution can be pipetted with standard pipettes, which demonstrates the existence of DNA in the lysate. If copolymer concentrations of 0.01% to 0.05% are used with ten minutes incubation time, cytoplasmatic proteins are detectable via SDS-PAGE, and there is no increase of viscosity. So, by using lower polymer concentrations a membrane solubilization can be achieved while the nucleus stays intact.

Claims

Claims Method for lysing cells and tissue, wherein the cells and / or the tissue are brought into contact with a polymer. The method according to claim 1, wherein the cell is an eucaryotic cell. The method according to any of the preceding claims, wherein the polymer is a polymer that can solubilize and stabilize membrane proteins and GPCRs. The method of claim 3, wherein said polymer has hydrophilic groups, such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholines, and hydrophobic groups, such as polymerized styrene groups, polymerized diisobutylene groups, or linear Cl to Cl 6 aliphatic group, branched Cl to Cl 6 aliphatic group, and cyclic C5 to C12 aliphatic group or C5 to C12 aromatic group. The method according to any of claims 3 to 5, wherein the polymer is selected from the group consisting of polyacrylic acid, optionally with a part of the carboxylic groups are coupled with cyclic amines to amides, diisobutylene / maleic acid copolymer, styrene / maleic acid copolymer, and acrylic acid / styrene copolymer. The method of claims 3 to 5, wherein the molecular weight of the polymer is 1900 to 20000. The method according to any of the preceding claims, wherein the concentration of the polymer is 0.01 % to 5 %. The method of any of the preceding claims, wherein the cell and / or the tissue is brought into contact with the polymer during less than 5 minutes. The method of any of the preceding claims, wherein the cell and / or the tissue is brought into contact with the polymer during less than 1 minute. The method of any of the preceding claims, wherein the cell and / or the tissue is brought into contact with the polymer during less than 30 secondsThe method of any of the preceding claims, wherein the method is employed for separating eucaryotic cells or cell components from procaryotic cells or cell components. The method of any of the preceding claims, wherein the method is employed for perforation of cell walls, leaving the cell cores intact. The method of any of the preceding claims, wherein the method is employed for perforation of cell walls, leaving the biggest part of the cells alive. The method of any of the preceding claims, wherein the method is employed for the introduction of foreign DNA or RNA into human, animal and other eukaryotic cells. Use of the polymer as defined in any of claims 3 to 6 for totally or partially lysing cells and tissue. Use of the polymer as defined in any of claims 3 to 6 for lysing muscle tissue, heart tissue, brain tissue, liver tissue, kidney tissue, lung tissue, or blood cells. Use of the polymers as defined in any of claims 3 to 6 in nucleic acid sequencing procedures. Use of the polymers as defined in any of claims 3 to 6 in long read sequencing procedures. Use of the polymer as defined in any of the claims 3 to 6 for the preparation of stable and active membrane proteins. Use of the polymer as defined in any of the claims 3 to 6 for the preparation of active soluble proteins. Use of the polymers as defined in any of the claims 3 to 6 in proteomics. Use of the method of any of claims 1-14 in drug discovery and expression proteomics. Use of the method of any of claims 1-14 for next sequencing applications. A kit for cell lysis, containing one or more polymers of any of the preceding claims.A kit for cell lysis of claim 24, also containing one or more substances from the following list:-a reductant, such as DTT (dithiothreitol) E-mercaptoethanol, or TCEP ((tris(2- carboxyethyl)phosphine)-a buffer component, such as HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), MES 2-(N-morpholino)ethanesulfonic acid, Tris [tris(hydroxymethyl)aminomethane], or Bis-Tris 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-l,3-diol]-a chelator, such as EDTA (ethylene diamine tetraacetic acid), EGTA (ethylene glycol-bis(P- aminoethyl ether)-N,N,N',N'-tetraacetic acid), NTA (nitrilotriacetic acid), or DTPA (diethylenetriaminepentaacetic acid)-one or more components from the group of lithium- sodium, or potassium salts.

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