阅读说明：本技术 用于从生物样品、优选加工过的植物籽粕分离膜结合的蛋白质的方法和手段 (Methods and means for separating membrane-bound proteins from biological samples, preferably processed plant seed meal ) 是由 M·K·威瑞克 M·夏 J·斯莱萨列娃 于 2018-12-12 设计创作，主要内容包括：本发明提供了从生物样品中,例如在来自粗的或加工过的植物材料、优选脱脂的植物籽粕例如卡诺拉油菜籽粕的样品中有效分离膜结合蛋白的方法和手段。所述生物样品可以被高度加工,例如通过施加高温、压力或化学处理,并且可以来自种子基质以及其他典型的植物组织,例如种子、谷粒、叶片、根或花粉。本发明包括提供新的提取缓冲液(MEB)以及其在本发明方法中的应用,其中所述缓冲液具有10-12.5的强碱性pH并且包含0.5％-5％水平的可溶浓度的去污剂。(The present invention provides methods and means for efficiently separating membrane-associated proteins from biological samples, for example, samples derived from crude or processed plant material, preferably defatted plant seed meal such as canola meal. The biological sample may be highly processed, for example by application of heat, pressure or chemical treatment, and may be from seed substrates as well as other typical plant tissues, such as seeds, grains, leaves, roots or pollen. The present invention comprises providing a novel extraction buffer (MEB) and its use in the method of the invention, wherein the buffer has a strongly alkaline pH of 10-12.5 and comprises a soluble concentration of detergent at a level of 0.5-5%.)

1. A method for extracting membrane-bound proteins from biological material, comprising: samples were incubated in a buffer at pH10 to 12.5 containing a soluble concentration of 0.5% to 5% of detergent (membrane extraction buffer (MEB)).

2. The method according to claim 1, which uses the lysis buffer MEB at a pH of 11.5 to 12.5.

3. The method of claim 1, wherein the sample is incubated at an incubation temperature of 35 ℃ to 55 ℃.

4. A method according to any one of claims 1-3, comprising applying a lysis buffer MEB containing a soluble concentration of ionic detergent of 1.5-5%.

5. The method according to any one of claims 1-4, comprising applying a lysis buffer MEB containing 1.5-3% LDS.

6. The method according to any one of claims 1-5, wherein the lysis buffer MEB comprises a reducing agent.

7. The method of claim 6, wherein the lysis buffer MEB comprises DTT, BME or TCEP-HCL.

8. The method according to claim 6 or 7, wherein the lysis buffer MEB comprises one or more reducing agents equivalent to 20mM-70 mtcep.

9. The method of any one of claims 1-8, wherein the MEB buffer comprises 20mM-70mM TCEP or 30-120nM DTT or 1-5% BME.

10. The method of any one of claims 1-9, comprising a pH-dependent buffer or salt solution.

11. The method of claim 10, wherein the buffer is Tris-CL, HEPES, CAPS, and/or sodium phosphate buffer NaH2PO4/Na2HPO 4.

12. The method of claim 10 or 11, wherein the buffer is:

i.5-300mM Tris-CL，

ii.20-40mM HEPES，

iii 10-40mM sodium phosphate (NaH2PO4/Na2HPO4), and/or

iv.50mM-500mM CAPS。

13. The method of any one of claims 1-12, comprising a viscosity agent.

14. The method of any one of claims 1-13, comprising a viscosity agent, wherein the viscosity agent is 10% -25% glycerol.

15. The method according to any one of claims 1-14, comprising: a buffer of pH10 to 12.5, wherein the buffer is 100mM to 150mM Tris-Cl, preferably 120mM, LDS content is 1.5% to 2.5%, reducing agent is 20 to 70mM TCP, preferably 50mM, and 10% to 20% glycerol.

16. The method of any one of claims 1-15, wherein the incubation time at the incubation temperature is from 10min to 180 min.

17. The method according to any one of claims 1-16, wherein extraction is performed as a plurality of sequential extractions, preferably said extraction is repeated 3 times or less, even more preferably said extraction occurs 2 times or less, most preferably only once.

18. The method of any one of claims 1-17, wherein the tissue is from a plant sample.

19. The method of any one of claims 1-17, wherein the tissue is from a leaf, seed, grain, leaf, root, or pollen.

20. The method of any one of claims 1-19, wherein the material is defatted plant seed meal.

21. The method according to any one of claims 1-20, comprising the steps of:

a. incubating the sample in a lysis buffer MEB according to any of claims 1-15 at a temperature of 35 ℃ to 55 ℃, which buffer comprises a detergent and optionally a reducing agent,

b. the membrane-bound protein is extracted,

c. the extract was diluted to adjust pH for protein analysis,

d. the protein is analyzed without prior precipitation, resuspension or solubilization.

22. The method of any one of claims 1-21, wherein the extracted protein is analyzed in western blot, mass spectrometry, isoelectric focusing, or 2D protein separation.

23. A composition comprising a buffer at pH 10-12.5 and a detergent in an amount of 0.5% -2.5%, and a reducing agent.

24. The composition of claim 23, comprising a pH of between pH10.0 and pH12.5

i.1.5% -2.5% LDS, and

ii.30mM-70mM TCEP.

25. The composition of claim 23 or 24, further comprising tissue and/or a membrane from an organism.

26. The composition of claim 23 or 25, having a temperature of 35 ℃ -55 ℃.

27. The method according to any one of claims 1-21, comprising one or more of the following steps:

a. the sample is ground and the sample is ground,

b. the sample was cooled on dry ice and,

c. the sample is weighed and the weight of the sample,

d. extracting the polypeptide from the ground and cooled sample by applying an extraction buffer MEB for use in the method of any one of claims 1-21 or a composition of any one of claims 23-26, and

e. the sample was centrifuged and the supernatant was transferred to a new tube, thereby cooling the tube.

28. The method of any of claims 1-22 or 27, comprising:

a. providing a plant meal, wherein the plant meal is prepared by mixing plant meal,

b. adding a pre-heated extraction buffer MEB for use in the method according to any one of claims 1 to 21 or a composition according to any one of claims 23 to 26,

c. the sample is ground and the sample is ground,

d. the samples were incubated at 30 ℃ to 45 ℃ for 5 to 15 minutes,

e. centrifuging the sample and transferring the supernatant to a new container, and

f. the supernatant was analyzed.

29. The method of any of claims 1-2 and 27 or 28, comprising: the one or more polypeptides are quantitatively analyzed by applying one or more antibodies that specifically bind to the potential membrane-bound polypeptides.

30. The method according to any one of claims 1-9 and 16-18, wherein the polypeptide is a desaturase or elongase, preferably a transgenic desaturase or elongase.

The methods described in the prior art are precipitation methods, which require resuspension and/or solubilization prior to immunoassay analysis.

Therefore, there is a need for a new extraction method to extract membrane proteins, for example from highly processed meals in an efficient manner, but allowing for immediate analysis of the proteins.

Accordingly, the present invention provides a method of extracting membrane-bound proteins from biological material, comprising:

the sample is incubated at an incubation temperature of 35 ℃ to 55 ℃ in a buffer of pH10 to 12.5, the buffer comprising 0.5% to 3% detergent.

Elevated temperatures, harsh denaturants and/or detergents were found to result in efficient extraction of membrane bound proteins. Thus, as a result of the need for an extraction method suitable for immediate analysis, new extraction buffer formulations and optimized extraction conditions were developed to allow efficient extraction of membrane-bound proteins from oilseed meals such as canola meal.

The use of a combination of detergent and high pH is required to ensure high extraction rates of membrane-bound proteins from highly processed plant seed substrates. In addition, the extraction may be performed at warm temperatures exceeding 30 ℃ to solubilize the buffer components and prevent aggregation of the extracted membrane proteins.

The use of this method in combination with a reducing agent can achieve very high percent protein extraction, e.g., up to 100%, and can successfully quantify the levels of protein extracted from high oilseed meal, e.g., canola meal, or other highly processed plant seed substrates, as well as other typical plant tissues, e.g., seeds, grains, leaves, roots, or pollen.

In addition, the method of the invention is suitable for analysis immediately after dilution to adjust or neutralize the pH for the analysis step, preferably without prior resuspension and/or dissolution of the sample.

The term "membrane protein" or "membrane-bound protein" encompasses all proteins associated with a biological membrane or matrix, including but not limited to proteins in the membrane of a cell, such as polypeptides, for example in the membrane of a cell wall or organelle, including but not limited to, for example, transmembrane proteins and peripheral membrane proteins; the proteins may be linked in different ways, such as but not limited to proteins associated with the seed matrix and other typical plant tissues such as grain, leaf, root or pollen.

It was found that extraction at warm temperatures improved the solubility of the buffer components while preventing aggregation of the extracted membrane proteins. The combination of detergents such as LDS, high pH and high temperature, enables efficient extraction. In combination with a reducing agent such as TCEP, very high, usually complete, extraction of membrane-bound proteins from canola seed meal can be achieved.

Using this method and achieving a high percentage of protein extraction, it is possible to successfully quantify the level of protein extracted from plant seed meal. Thus, the methods of the invention and the buffers of the invention allow for the successful extraction of insoluble and soluble proteins from other highly processed plant seed substrates as well as other typical plant tissues such as seeds, grains, leaves, roots or pollen.

Thus, in the methods of the invention, a new combination of lysis buffer components and extraction conditions identified as effective for extracting membrane bound proteins uses an optimized membrane bound protein lysis buffer (referred to herein as "membrane extraction buffer" or "MEB") that contains a high concentration of detergent, e.g., 0.5% to 5%, at a high pH, e.g., pH10 to pH 12.0.

When the preparation is used for extraction at high incubation temperatures, for example at 35 ℃ to 55 ℃, the membrane bound protein can be efficiently extracted and the resulting clarified extract is suitable for immediate analysis, for example by immunoblotting after dilution of the extract to neutral pH. The extracted proteins can be analyzed, for example, by mass spectrometry, isoelectric focusing, 2D gel separation of proteins, proteomic analysis, or western blotting.

Thus, the method of the invention comprises incubating the sample in an MEB buffer comprising a detergent content of 0.5 or 1.5% to 5%, more preferably 1.5% to 3%, most preferably about 2%. The concentration of detergent depends on the temperature and solubility of the detergent. Thus, the detergent and its content are selected according to the exact temperature and pH used in the method of the invention. Soluble concentrations of detergent are used in the methods of the invention.

Thus, a method for extracting membrane bound proteins from biological material comprises incubating a sample in a lysis buffer at a pH of 10 to 12.5, the lysis buffer comprising a soluble concentration of 1.5% to 5% of a detergent.

Furthermore, the present invention relates to a method for extracting membrane-bound proteins from biological material, comprising incubating a sample in a lysis buffer MEB of pH10 to 12.5 at an incubation temperature of 35 ℃ to 55 ℃, said lysis buffer comprising a detergent in a soluble concentration of 0.5% to 5%.

The detergent may be an ionic or non-ionic detergent depending on the type of protein and tissue used in the sample. Examples of other detergents are TrintonX-100, NP40, C7bzO, CHAPS, sodium deoxycholate, LDS or SDS, for example as described in http:// www.vbcf.ac.at/filmpin/user _ upload/ProTech/SW _ Buffer _ compatibility.

TritonX-100 is used, for example, at 0.5% to 3% in lysis buffer. LysoFos Choline12 is used, for example, in a concentration of 0.5% to 10% in lysis buffer. NP40 is used, for example, at 0.5% to 1% in lysis buffer. C7Bzo is used, for example, at 0.5% to 2% in lysis buffer. CHAPS is used, for example, at 0.5% to 2% in lysis buffer. Sodium deoxycholate is used, for example, at 0.5% to 2% in lysis buffer. SDS is used, for example, at 0.5% to 2% in lysis buffer.

The detergent may be an ionic detergent, such as SDS or LDS. It is known in the art that, for example, SDS has a lower solubility at high pH and low temperature than LDS. It has been found that LDS is well soluble for the temperature of the process of the invention. Thus, the method of the invention comprises incubating the sample in a MEB buffer comprising a detergent content of 1 to 3% LDS, e.g. 1.5% to 2.5% LDS, e.g. about 2% LDS.

The incubation temperature chosen for incubation in the method of the invention allows harsh extraction conditions, but requires that the proteins do not aggregate. Thus, the incubation is carried out at an incubation temperature of more than 30 ℃, for example an incubation temperature of more than 35 ℃, such as 37 ℃ or more, 40 ℃ or more, 42 ℃ or more, 45 ℃ or more, 50 ℃ or more, 55 ℃ or more, but less than 75 ℃, even more preferably less than 70 ℃, such as 65 ℃ or less, 60 ℃ or less, 55 ℃ or less, 50 ℃ or less, 47 ℃ or less, 45 ℃ or less, 42 ℃ or less, such as about 35 ℃, about 37 ℃, about 40 ℃, or about 42 ℃, or about 45 ℃, or about 47 °, or about 50 ℃, or about 60 ℃. Thus, the incubation of the method of the invention may be carried out between 35 ℃ and 55 ℃, or between 37 ℃ and 50 ℃ or between 40 ℃ and 47 ℃.

There is a need for extraction methods suitable for immediate analysis, such as western blot analysis, and therefore a new extraction buffer formulation has been developed which, in combination with optimized extraction conditions, can efficiently extract membrane-bound proteins from canola oil seed meal. This method allows for the immediate analysis of proteins using western blotting methods, e.g. only after neutralisation of the buffer pH, e.g. by diluting the sample.

Furthermore, the lysis buffer used during the incubation step of the method of the invention comprises a reducing agent. Reducing agents are well known in the art, for example DTT (DL-dithiothreitol; Clelans reagent z), BME (. beta. -mercaptoethanol) or TCEP-HCL (tris (2-carboxyethyl) phosphine hydrochloride). In standard lysis buffers, the concentration of DTT ranges from 5 to 50mM, BME from 200mM to 1M, and TCEP from 0.5 to 10 mM.

If the same reducing power as 5mM to 100mM TCEP-HCL is produced, one reducing agent or a mixture of reducing agents may be used. Preferably, the reducing power corresponds to, for example, a reducing power of 20mM to 70mM TCEP-HCL, 40mM to 60mM TCEP-HCL, e.g., about 50mM TCEP-HCL.

The MEB lysis buffer used in the incubation of the method of the invention comprises, for example, 5mM to 100mM TCEP, e.g.20 mM to 70mM TCEP-HCL, 40mM to 60mM TCEP, e.g.about 50mM TCEP. Thus, an MEB lysis buffer, for example, comprises 20mM TCEP or greater, 30mM TCEP or greater, 40mM TCEP or greater, and 100mM TCEP or less, 80mM TCEP or less, 70mM TCEP or less, or 60mM TCEP or less. In one embodiment, the TCEP is present in an amount of about 50 mM.

The MEB lysis buffer used in the methods of the invention may also comprise, for example, 30 to 120nM DTT, for example 50nM to 100nM DTT.

MEB lysis buffer used in the methods of the invention may also comprise, for example, 1-5% BME, e.g., 2% -4% BME.

The combination of LDS as an ionic detergent and TCEP as a reducing agent allows almost complete extraction of membrane-bound proteins from canola meal at high pH values (e.g., at about pH 12.0). The ability to perform the extraction at warm temperatures helps maintain the solubility of the buffer components while preventing aggregation of the extracted membrane proteins.

Using this method, very high protein extraction rates, e.g., up to 100%, can be achieved, allowing successful quantification of protein levels extracted from high oil seed meal, e.g., from canola or other highly processed plant seed substrates, as well as other typical plant tissues such as seeds, grains, leaves, roots, or pollen.

The membrane extraction buffer MEB may comprise a suitable pH-dependent buffer, or a salt solution, or an aqueous solution, e.g. water. In addition, the buffer component may also be omitted. Standard buffers for lysis buffers are e.g.Tris-CL, HEPES, CAPS or sodium phosphate buffer NaH2PO4/Na2HPO 4. Typically, the concentration of the buffer is: 5 to 300mM Tris-CL, 20 to 40mM HEPES, 10 to 40mM sodium phosphate (NaH2PO4/Na2HPO4), or 50mM to 500mM CAPS.

Thus, the method of the invention may comprise: incubate in lysis buffer MEB with or without buffer.

The pH of the buffer used in the incubation is between 10 and 12.5, for example between pH11 and pH12.5, for example around pH12.

Preferably, the lysis buffer MEB comprises 5 to 300mM Tris-CL, such as about 110mM, about 150mM, about 200mM or about 250mM, such as less than 150mM, such as 140mM or less, such as 50 to 250mM, 70 to 200mM, 100 to 150 mM.

In one embodiment, the lysis buffer MEB used in the method of the invention further comprises a viscosity agent, such as glycerol or sucrose. For example, the lysis buffer MEB used in the method of the invention comprises 5% to 30% glycerol, preferably 10% to 25% glycerol, for example about 20% glycerol.

Thus, the method of the invention comprises incubating a sample comprising a membrane protein in a lysis buffer MEB comprising:

a buffer having a pH of from 10 to 12.5, and a detergent content of from 0.5% to 2.5%, or

A buffer having a pH of from 10 to 12.5, and a detergent content of from 0.5% to 3%, preferably LDS or SDS, and a viscosity agent, or

A buffer having a pH of from 10 to 12.5, an LDS content of from 1.5% to 2.5%, preferably about 2%, and a viscosity agent, or

A buffer having a pH of from 10 to 12.5, LDS in an amount of from 1.5% to 2.5%, preferably about 2%, and a reducing agent, or

A buffer having a pH of from 10 to 12.5, an LDS content of from 1.5 to 2.5%, preferably about 2%, and a reducing agent and a viscosity agent, or

A buffer having a pH of from 10 to 12.5 and an LDS content of from 1.5% to 2.5%, preferably about 2%; reducing agent is TCEP in the range of 20 to 70mM, preferably 50mM, and viscosity agent glycerol, preferably in the range of 15 to 25%, or

A buffer, wherein the buffer is 100mM to 150mM Tris-Cl, preferably 120mM, at pH10 to 12.5, LDS content 1.5% to 2.5%, reducing agent is 20 to 70mM TCEP, preferably 50mM, and 10% to 20% glycerol, or

A buffer, wherein the buffer is about 120mM Tris-CL, pH is about 12, LDS content is about 2%, reducing agent is about 50mM TCEP, and glycerol is about 20%.

The incubation during the method of the invention is carried out for a suitable period of time: if too short, the extraction efficiency will be affected, and if too long, the protein will be affected by aggregation. The incubation time at the incubation temperature is for example between 2 minutes and 180 minutes, for example between 5 minutes and 60 minutes, for example about 5, 10 or 15 minutes.

The extraction is performed as a single or multiple sequential extractions, preferably the extraction is repeated 3 times or less, even more preferably the extraction occurs 2 times or less, most preferably only once.

The method of the invention allows for efficient extraction of membrane-bound proteins from biological samples, such as plants or parts or fractions of plants, such as seeds, grains, leaves, roots or pollen or cell samples. The process of the invention particularly allows for efficient extraction from a meal of plant origin, for example from an oilseed plant seed meal, a Brassica seed meal, for example a Brassica napus (Brassica napus) seed meal or a canola seed meal, or from a soybean meal. A method for producing Canola oil seed Meal is described in Donna M.Klockeman, Romeo Toledo and KevinA.Sims.isolation and Characterization of degraded Canola Meal Protein (1997). J.Agric.food chem.45: 3867-3870.

The content of the seed meal in the buffer may be 1% or more, such as 5% or more, 10% or more, 15% or more, 17% or more, 20% or more, 25% or more, but less than 60%, such as 50%, 30%, 25% or less. Thus, in the process of the invention, the content of meal may for example be between 2% and 50%, such as 5 to 40%, such as 10% to 20%, or 5% to 15%.

The meal is, for example, meal from a plant, for example, seed meal from an oilseed plant such as brassica species, in particular canola. Meal such as canola oil seed meal is a by-product of canola oil production.

Thus, in one embodiment, the proteins comprised in the sample are denatured. The production of seed meal, such as seed oil from high oil plants, such as seed oil from canola, often requires the use of harsh processing conditions. The resulting seed meal contains denatured protein. The methods of the invention also include the use of denatured protein samples, e.g., samples in which substantially all of the protein is denatured. For example, at least 30% or 50% of the protein is denatured, or at least 60%, 70% or 90% or about 100% is denatured.

The lysis buffer may also comprise a viscosity agent. Standard viscosity agents are glycerol and sucrose. Thus, the buffer used in the incubation step of the method of the invention may comprise 5% to 25% glycerol, for example 10% to 20%, preferably about 20% glycerol.

Thus, in one embodiment, the Meal Extraction Buffer (MEB) consists of 120mM Tris, 20% glycerol, 2% LDS and 50mM TCEP, at a pH of 12.0 and a temperature of about 40 ℃. To extract the protein, it is added to canola meal and incubated with mixing at 40 ℃ for 5 to 15 minutes. The crude extract may then be clarified, for example by spin centrifugation, and the supernatant further analyzed.

The clarified extract obtained by the method of the invention is suitable for immediate analysis, for example by mass spectrometry, isoelectric focusing or 2D protein separation, western blot analysis. Thus, in one embodiment, the method of the invention comprises the further steps of: the sample is centrifuged, for example in a centrifuge, and the supernatant is transferred to a new tube, preferably the tube is cooled thereby.

Accordingly, the present method also relates to a method comprising: incubation of samples as described above, clarification of the extracts from the incubation, followed by analysis of the extracted proteins by ELISA, western blot, mass spectrometry, isoelectric focusing or 2D protein separation, protein sequencing.

Thus, in one embodiment, the method of the invention comprises the steps of:

a. the sample is ground and the sample is ground,

b. the sample is cooled, for example on dry ice,

c. the sample is weighed and the weight of the sample,

d. extracting polypeptides from the ground and cooled sample by applying the extraction buffer MEB of the invention or said buffer MEB for use in the method of the invention and incubating the sample at the incubation temperature of the invention, and

e. the sample was centrifuged and the supernatant was transferred to a new tube, thereby cooling the tube.

Thus, in one embodiment, the method of the invention comprises the steps of:

a. providing a plant seed meal, wherein the plant seed meal,

b. adding a pre-heated extraction buffer MEB according to the invention or said buffer MEB for use in a method according to the invention to the plant material,

c. the sample is ground and the sample is ground,

d. incubating the sample at 30 ℃ to 45 ℃, preferably at about 40 ℃ for 1 to 15 minutes, preferably 2 to 5 minutes, more preferably about 3 minutes,

e. the sample was centrifuged and the supernatant was transferred to a new container,

f. the proteins in the supernatant are analyzed, for example, by western blotting, mass spectrometry, isoelectric focusing or 2D protein separation.

The method of the invention may then comprise the following further steps: the one or more polypeptides, such as desaturases or elongases (elongases), preferably transgenic desaturases or elongases, are quantitatively assayed by the use of one or more antibodies that specifically bind to the potential membrane-bound polypeptide.

Furthermore, the invention relates to a composition for extracting proteins. Accordingly, a novel Meal Extraction Buffer (MEB) is identified herein to efficiently extract membrane-bound proteins, such as proteins from plant seed meal. Thus, the present invention also relates to a novel lysis buffer MEB for efficient extraction of membrane bound proteins, as described above for the method of the invention.

The new Meal Extraction Buffer (MEB) has a pH between 10 and 12.5, e.g., between pH11 and pH12.5, e.g., about pH12.

MEB lysis buffer comprises a detergent content of 0.5 or 1.5% to 5%, more preferably 1.5% to 3%, most preferably about 2%. The concentration of detergent depends on the temperature and solubility of the detergent. Thus, the detergent and its content are selected according to the exact temperature and pH used in the method of the invention. Soluble concentrations of detergent are used in the methods of the invention.

The detergent may be an ionic or non-ionic detergent depending on the type of protein and tissue used in the sample. Examples of other detergents are TrintonX-100, NP40, C7bzO, CHAPS, sodium deoxycholate, LDS or SDS, for example as described by http:// www.vbcf.ac.at/filmadin/user _ upload/ProTech/SW _ Buffer _ compatibility.

TritonX-100 is used, for example, in a lysis buffer at a concentration of 0.5% to 3%. LysoFos Choline12 is used, for example, in a concentration of 0.5% to 10% in lysis buffer. NP40 is used, for example, at 0.5% to 1% in lysis buffer. C7Bzo is used, for example, at 0.5% to 2% in lysis buffer. CHAPS is used, for example, at 0.5% to 2% in lysis buffer. Sodium deoxycholate is used, for example, at 0.5% to 2% in lysis buffer. SDS is used, for example, at 0.5% to 2% in lysis buffer.

The detergent may be an ionic detergent, such as SDS or LDS. It is known in the art that, for example, SDS has a lower solubility at high pH and low temperature than LDS. It has been found that LDS is well soluble for the temperature of the process of the invention. Thus, the MEB buffer comprises a detergent content of 1 to 3% LDS, e.g. 1.5% to 2.5% LDS, e.g. about 2% LDS.

The MEB lysis buffer comprises, for example, a reducing agent or mixture of reducing agents, which can be used if the same reducing power as 5mM to 100mM tcp cl is produced. Preferably, the reducing power corresponds to, for example, a reducing power of 20mM to 70mM TCEP-HCL, 40mM to 60mM TCEP-HCL, e.g., about 50mM TCEP-HCL.

In one embodiment, the MEB lysis buffer comprises 5mM to 100mM TCEP, e.g., 20mM to 70mM TCEP-HCL, 40mM to 60mM TCEP, e.g., about 50mM TCEP. Thus, for example, the MEB lysis buffer comprises 20mM TCEP or greater, 30mM TCEP or greater, 40mM TCEP or greater, and 100mM TCEP or less, 80mM TCEP or less, 70mM TCEP or less, or 60mM TCEP or less. In one embodiment, the TCEP is present in an amount of about 50 mM.

The MEB lysis buffer comprises, for example, 30 to 120nM DTT, for example, 50nM to 100nM DTT.

The MEB lysis buffer may also comprise, for example, 1-5% BME, e.g., 2% -4% BME.

In one embodiment, the membrane extraction buffer MEB comprises a suitable pH-dependent buffer, or a salt solution, or an aqueous solution, e.g. water. In addition, the buffer component may also be omitted. Standard buffers for lysis buffers are e.g.Tris-CL, HEPES, CAPS or sodium phosphate buffer NaH2PO4/Na2HPO 4. Typically, the concentration of the buffer is: 5 to 300mM Tris-CL, 20 to 40mM HEPES, 10 to 40mM sodium phosphate (NaH2PO4/Na2HPO4), or 50mM to 500mM CAPS.

In one embodiment, the lysis buffer MEB further comprises a viscosity agent, such as glycerol or sucrose. For example, lysis buffer MEB comprises 5% to 30% glycerol, preferably 10% to 25% glycerol, e.g. about 20% glycerol.

Thus, the new lysis buffer MEB comprises, for example:

a buffer having a pH of from 10 to 12.5, and a detergent content of from 0.5% to 2.5%, or

A buffer having a pH of from 10 to 12.5, and a detergent content of from 0.5% to 3%, preferably LDS or SDS, and a viscosity agent, or

A buffer having a pH of from 10 to 12.5, an LDS content of from 1.5% to 2.5%, preferably about 2%, and a viscosity agent, or

A buffer having a pH of from 10 to 12.5, LDS in an amount of from 1.5% to 2.5%, preferably about 2%, and a reducing agent, or

A buffer having a pH of from 10 to 12.5, an LDS content of from 1.5 to 2.5%, preferably about 2%, and a reducing agent and a viscosity agent, or

A buffer having a pH of from 10 to 12.5, an LDS content of from 1.5% to 2.5%, preferably about 2%, a reducing agent of from 20 to 70mM of TCEP, preferably 50mM, and a viscosity agent of glycerol, preferably from 15 to 25%, or

A buffer, wherein the buffer is 100mM to 150mM Tris-Cl, preferably 120mM, at pH10 to 12.5, LDS content 1.5% to 2.5%, reducing agent is 20 to 70mM TCEP, preferably 50mM, and 10% to 20% glycerol, or

A buffer, wherein the buffer is about 120mM Tris-CL, pH is about 12, LDS content is about 2%, reducing agent is about 50mM TCEP, and glycerol is about 20%.

In one embodiment, the temperature of the buffer MEB is higher than 35 ℃, such as 37 ℃ or higher, 40 ℃ or higher, 42 ℃ or higher, 45 ℃ or higher, 50 ℃ or higher, 55 ℃ or higher, but lower than 75 ℃, even more preferably lower than 70 ℃, such as 65 ℃ or lower, 60 ℃ or lower, 55 ℃ or lower, 50 ℃ or lower, 47 ℃ or lower, 45 ℃ or lower, 42 ℃ or lower, such as about 35 ℃, about 37 ℃, about 40 ℃, or about 42 ℃, or about 45 ℃, or about 47 ℃, or about 50 ℃, or about 60 ℃. Thus, the temperature of the buffer is for example between 35 ℃ and 55 ℃, or between 37 ℃ and 50 ℃, or between 40 ℃ and 47 ℃.

In a further embodiment, the lysis buffer MEB also comprises seed meal, e.g. 1% or more, such as 5% or more, 10% or more, 15% or more, 17% or more, 20% or more, 25% or more, but less than 60%, e.g. 50%, 30%, 25% or less. Thus, in the process of the invention, the content of meal may for example be between 2% and 50%, such as 5 to 40%, such as 10% to 20%, or 5% to 15%.

Furthermore, the lysis buffer MEB comprises biological material, e.g. from a plant or a part or fraction of a plant, such as seeds, grains, leaves, roots or pollen, or a cell sample, e.g. an oilseed plant seed meal, such as a seed meal from a Brassica species, such as Brassica napus (Brassica napus) or canola seed meal, or soybean meal.

Reference to the literature

[1]Canola Council of Canada.Steps in Oil and Meal Processing.

https：//www.canolacouncil.org/oil-and-meal/what-is-canola/how-canola-is-processed/steps-in-oil-and-meal-processing/#Pressing.(Accessed 8Nov 2017).

[2]Donna M.Klockeman，Romeo Toledo，and Kevin A.Sims.Isolation andCharacterization of Defatted Canola Meal Protein.(1997).J.Agric.Food Chem.45：3867-3870.

Examples