CN114487070B

CN114487070B - Kit for identifying protein purity and application thereof

Info

Publication number: CN114487070B
Application number: CN202011251470.7A
Authority: CN
Inventors: 柯天一; 张鹏飞; 劳芳; 孙雯倩; 李波; 姚德惠; 欧阳芳幸; 范墨林
Original assignee: Kunshan Xinyunda Biotech Co ltd
Current assignee: Kunshan Xinyunda Biotech Co ltd
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2024-04-19
Anticipated expiration: 2040-11-11
Also published as: CN114487070A

Abstract

The invention relates to a kit for identifying protein purity and application thereof. The invention provides an identification detection method of purified protein, which comprises the following steps: (1) pretreatment of purified protein: (2) purified protein identification detection. The protein pretreated by the method has fewer mixed proteins, the detection and evaluation results of the protein purity are more accurate, the protein purification effect is reflected more objectively, and more accurate basis is provided for the subsequent protein control. The invention can be operated at room temperature without freezing or high temperature, and without any experimental equipment; the operation is simple and convenient, the time consumption is short, and the rapid treatment can be completed in a kit mode; the method is applicable to two protein detection experiments of CE-SDS and SDS-PAGE; is applicable to various protein types and has universality.

Description

Kit for identifying protein purity and application thereof

Technical Field

The invention belongs to the field of biological macromolecule detection methods, and particularly relates to a kit for identifying protein purity and application thereof.

Background

Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are electrophoresis methods for detecting protein purity, and are recorded in pharmacopoeias, and the two methods are common technical means for product quality control in the process of drug development and are most conventional experimental methods for determining the purity and molecular weight of protein biomacromolecules such as fusion proteins, antibodies and the like.

SDS is an anionic surfactant, and after SDS is added into protein solution, SDS can make the hydrogen bond and hydrophobic bond of protein open and bind to protein molecule, so that it can be combined with protein to make protein conformational change. The shape of the protein after binding to SDS in aqueous solution is similar to cigar-shaped oblong bars. The difference in charge and the resulting different mobilities of different protein molecules separate the proteins. If the protein sample contains a plurality of proteins or the purified protein sample contains other hetero proteins, the different proteins are separated into a plurality of protein bands after SDS-PAGE separation. If the purified sample contains only the same protein, only one protein band will appear after electrophoresis of the protein sample. Whereby the CE-SDS and SDS-PAGE techniques allow analysis of the purity of the protein samples.

The principle of CE-SDS and SDS-PAGE is the same, and proteins are separated according to the molecular weight. In the prior art, before SDS-PAGE and CE-SDS are carried out to detect the purity of the protein, a sample is generally pretreated by adopting a high-temperature heating mode, so that the protein is denatured at the temperature of 95-98 ℃ and the protein with multiple subunits is dissociated into single subunits. If the subject is an immunoglobulin, a disulfide bond-containing protein, a reducing agent is usually added to open one disulfide bond in the protein (reduce disulfide bonds). The peptide chains in the pretreated sample are all in a disulfide-bond-free, isolated state. This conventional pretreatment is used in the prior art. However, the inventors of the present application have found by accident that the sample pretreatment method by high temperature heating causes the primary structure of the purified protein to be destroyed, thereby introducing a new hetero-protein having a molecular weight smaller than that of the target protein, and causing the CE-SDS and SDS-PAGE purity analysis maps to exhibit hetero-protein bands introduced by the high temperature pretreatment, so that the result of the protein purity detection and identification is not objective and inaccurate. It has also been reported that SDS-PAGE samples can be pretreated with trichloroacetic acid, but the pretreatment reagent has a large influence on the structural stability of proteins and can cause the aggregation or cleavage of protein molecules into fragments, so that the use of trichloroacetic acid as a protein precipitant interferes with the accuracy of the results of SDS-PAGE analysis of protein purity (influence of trichloroacetic acid on the structural stability of proteins, guo Lian et al, university of medical science, fourth force, 2001, 22 (22)).

Therefore, the incorrect information provided by the improper pretreatment mode can influence the protein purity analysis result, mislead the quality control strategy of the subsequent products and influence the patent medicine. However, so far, few related researches on improvement of a protein purity detection and identification method exist, and researches on pretreatment modes of CE-SDS or SDS-PAGE samples are not reported. In order to reduce or even avoid the occurrence of new impurities introduced during the detection of CE-SDS or SDS-PAGE, it is desirable to find a method for detecting and identifying purified proteins that replaces the high temperature heat pretreatment mode.

Disclosure of Invention

In order to solve the problem that the purity judgment is not affected by the introduction of the unreasonable hybrid protein in the high-temperature pretreatment method, the inventor conducts intensive research on sample pretreatment experimental methods of CE-SDS and SDS-PAGE under the motivation of searching for alternative better pretreatment methods, uses ferritin, BSA and IgG as samples to be tested, respectively represents multimeric globular proteins, general macromolecular proteins and immunoglobulins, explores various protein pretreatment methods, and compares the patterns of the CE-SDS and the SDS-PAGE in different pretreatment methods with the patterns of the conventional high-temperature treatment methods. The inventors creatively found that: the sample is pretreated by using low-concentration trifluoroacetic acid (TFA), so that the obtained CE-SDS and SDS-PAGE patterns have fewer impurity proteins, the purity state of the sample to be detected can be reflected more objectively, and a more scientific reference is provided for the subsequent product quality control scheme.

TFA is a strong carboxylic acid and is a good solvent for many organic compounds, such as carbon disulfide, which solubilizes proteins. TFA is often used as an ion pair reagent in reversed phase chromatography for the separation of polypeptides and proteins. In addition, higher concentrations of TFA are also acidic denaturing precipitants that can be used to precipitate proteins. However, it has not been found that it is possible to pretreat purified proteins prior to SDS-PAGE or CE-SDS instead of high temperature. The discovery of the application provides a more preferable technical scheme for solving the problem of sample purity detection analysis accuracy in the process of bio-macromolecule patent medicine.

In a first aspect, the invention relates to a method for identifying and detecting purified proteins, comprising:

(1) Pretreatment of purified protein: pretreating the purified protein with a low concentration of trifluoroacetic acid to obtain a pretreated purified protein, wherein the working concentration of the low concentration of trifluoroacetic acid is 0.01% -3% (v/v);

(2) And (3) identifying and detecting the purified protein: identifying and detecting the purity of the pretreated purified protein obtained in the step (1) by using sodium dodecyl sulfate capillary electrophoresis (CE-SDS) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In certain preferred embodiments, the low concentration trifluoroacetic acid operates at a concentration of 0.05% to 2% (v/v), preferably 0.1% to 1% (v/v); more preferably 0.5%.

In certain embodiments, the purified protein is a protein obtained by protein-induced expression by molecular biological methods, purification treatment, and subsequent use in SDS-PAGE, CE-SDS to detect purity after pretreatment by the methods of the present invention.

In a second aspect, the invention relates to the use of a method for pre-treating purified proteins with trifluoroacetic acid for the detection of protein purity, in particular for the detection of protein purity and/or the assessment of hybrid proteins by SDS-PAGE or CE-SDS methods.

In a third aspect, the invention relates to a kit for pre-treating purified protein with trifluoroacetic acid, wherein TFA is included in the kit, and the working concentration of TFA is 0.01% -3% (v/v).

Drawings

FIG. 1 shows SDS-PAGE detection of 0.1% TFA treated samples of recombinant human heavy chain ferritin (HFn) and samples treated at a high temperature of 95 ℃.

FIG. 2 shows a superposition of 3 Blank control (Blank) systems of recombinant human heavy chain ferritin.

FIG. 3 shows a CE-SDS overlay of recombinant human heavy chain ferritin in 0.1% TFA system and 95℃high temperature system.

Fig. 4 is a partial enlarged view of fig. 3.

FIG. 5 shows a CE-SDS overlay of recombinant human heavy chain ferritin treated in 0.1% TFA system for different times.

Fig. 6 is a partial enlarged view of fig. 5.

FIG. 7 shows CE-SDS overlays of recombinant human heavy chain ferritin treated with 0.1% TFA-10% ACN system at different times.

FIG. 8 shows a superposition of CE-SDS of recombinant human heavy chain ferritin treated in different systems (HFn-60 min, HFn-95-5min, 0.1% TFA-60min and 0.1% TFA-50% ACN-60 min).

Fig. 9 is an enlarged view of a portion of fig. 8.

FIG. 10 shows a CE-SDS overlay of recombinant human heavy chain ferritin treated at different ACN concentration systems for different times.

Fig. 11 is a partial enlarged view of fig. 10.

FIG. 12 shows the SDS-PAGE detection of recombinant human heavy chain ferritin fermentation supernatants.

FIG. 13 shows the SDS-PAGE (12% SDS-PAGE gel) results of recombinant human heavy chain ferritin treated with non-reducing loading buffer.

FIG. 14 shows the SDS-PAGE (12% SDS-PAGE gel) results of recombinant human heavy chain ferritin treated with a reduction loading buffer.

FIG. 15 shows the SDS-PAGE (12% SDS-PAGE gel) results of recombinant human heavy chain ferritin.

FIG. 16 shows the SDS-PAGE (10% SDS-PAGE gel) results of BSA and IgG.

FIG. 17 shows a superimposed graph of the CE-SDS assay of samples obtained after treatment of recombinant human heavy chain ferritin with TFA and SDS at various concentrations for various periods of time.

Fig. 18 is a partial enlarged view of fig. 17.

Detailed Description

I. Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification. The polypeptides described herein may be, for example, wild-type proteins, biologically active fragments of wild-type proteins, or variants of wild-type proteins or fragments. Variants of the invention may contain amino acid substitutions, deletions or insertions. Substitutions may be conservative or non-conservative. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. When the polypeptide comprises an amino acid mimetic or modified amino acid, the monomers may be linked by a bond other than peptide linkage or a derivative of peptide linkage.

As used herein, the term "purified" or "isolation" refers to a polypeptide that has been separated from or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) with which it is naturally associated (e.g., other proteins, lipids, and nucleic acids in cells expressing the protein). Typically, a polypeptide is purified when it comprises at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) wt% of the total protein in the sample.

As used herein, the term "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide).

As used herein, the term "contacting" refers to a process whereby at least two different substances (e.g., compounds or cells comprising biomolecules) become sufficiently adjacent to react, interact, or physically touch.

As used herein, the term "pretreatment" refers to a preparation process performed before performing an experimental operation, and for the purposes of the present invention, refers to a method of performing a preliminary operation on a sample to be tested before performing an experimental test on the purity of the sample to be tested, such as an SDS-PAGE or CE-SDS test, in order to allow the experimental operation of the purity test to be successfully completed and obtain an experimental result for analysis, and before performing the experimental test on the purity of the sample to be tested.

As used herein, the term "physiological buffer" refers to a class of buffer solutions suitable for use under physiological conditions, and refers to a mixed solution containing weak acids and their corresponding salts, weak bases and their corresponding salts, and acid salts of polybasic acids and their corresponding secondary salts, which can counteract and alleviate the influence of external strong acids or strong bases on the pH of the solution to some extent, thereby maintaining the pH of the solution relatively stable.

Detailed description of the preferred embodiments

(2) And (3) identifying and detecting the purified protein: identifying and detecting the purity of the pretreated purified protein obtained in the step (1) by using sodium dodecyl sulfate capillary electrophoresis (CE-SDS) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

In certain embodiments of the invention, the purified protein is a protein obtained by a molecular biological method, protein-induced expression, and purification treatment.

In certain preferred embodiments of the present invention, the low concentration trifluoroacetic acid operates at a concentration of 0.05% to 2% (v/v), preferably 0.1% to 1% (v/v); more preferably 0.5% (v/v).

In certain preferred embodiments of the invention, the concentration of protein in the purified protein is in the range of 0.2-20mg/ml, preferably 0.2-10mg/ml, preferably 0.5-5mg/ml, more preferably 0.75-5mg/ml.

In certain preferred embodiments of the invention, the purified protein is contacted with trifluoroacetic acid in a manner selected from the group consisting of: adding the purified protein to a solution containing trifluoroacetic acid, adding the purified protein and trifluoroacetic acid to the solution simultaneously, and adding trifluoroacetic acid to the solution of purified protein; purified protein was mixed with trifluoroacetic acid.

In certain preferred embodiments of the invention, after mixing the purified protein with trifluoroacetic acid, the protein is left at 4-25℃for more than 5min, preferably 5-60min, more preferably 5-30min.

In certain preferred embodiments of the invention, a protein reducing agent may also be added to the process.

In certain preferred embodiments of the present invention, the reducing agent is selected from one or more of beta-mercaptoethanol, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), or SDS.

In certain preferred embodiments of the invention, the reducing agent may be added to the solution system prior to, simultaneously with, or after contacting the protein with trifluoroacetic acid;

in certain preferred embodiments of the present invention, the concentration of said beta-mercaptoethanol is from 2 to 5% (v/v);

in certain preferred embodiments of the present invention, the Dithiothreitol (DTT) is 50-100mM;

In certain preferred embodiments of the present invention, the tris (2-carboxyethyl) phosphine (TCEP) is 5-50mM;

in certain preferred embodiments of the invention, the SDS is present in an amount of 1-3% (mass to volume).

In certain preferred embodiments of the present invention, acetonitrile (ACN) may also be added to the trifluoroacetic acid, wherein the working concentration of acetonitrile in the pretreatment system is 5% to 60% (v/v) and the working concentration of trifluoroacetic acid is 0.01% to 3% (v/v).

In certain preferred embodiments of the present invention, the working concentration of acetonitrile is from 10% to 50%; more preferably 15% -30%.

In certain preferred embodiments of the present invention, the trifluoroacetic acid has a working concentration of 0.05% to 2% (v/v), 0.1% to 1% (v/v); more preferably 0.5%.

In certain preferred embodiments of the invention, the solution is a physiological buffer or an aqueous solution.

In a third aspect the invention relates to a kit for pre-treating purified proteins with trifluoroacetic acid, said kit comprising TFA. Wherein the TFA may be provided in the form of a stock solution, a concentrate or the like, or in the form of a solution of a working fluid diluted to a working concentration, which working concentration in use is 0.01% to 3% (v/v), preferably 0.05% to 2%, more preferably 0.1% to 1%; more preferably 0.5%, the working solution is preferably a physiological buffer or aqueous solution of TFA. Optionally, the kit further comprises a reducing agent selected from the group consisting of beta-mercaptoethanol, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), SDS.

The invention has the technical effects that:

1. The protein pretreated by the method is less than the protein pretreated by high temperature, the protein purity detection and evaluation results are more accurate, the protein purification effect is reflected more objectively, and more accurate basis is provided for the subsequent protein control;

2. The method can be operated at room temperature, does not need to be carried out under freezing or high-temperature conditions, and does not need any experimental equipment;

3. the operation is simple and convenient, the time consumption is short, and the rapid treatment can be completed in a kit mode;

4. the method is applicable to two protein detection experiments of CE-SDS and SDS-PAGE;

5. is applicable to various protein types and has universality.

For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include some embodiments with a combination of all or some of the features described.

Examples

Materials and methods

1. Experimental instrument

Table 1 instrument

Name of the name	Manufacturer' s	Model number
			Small-sized high-speed refrigerated centrifuge	Thermo Fisher	Legend Micro 21R
Vortex instrument	Scientific Industries	Vortex-Genie 2
			Three-hole electric heating constant temperature water bath	Shanghai Yiheng	DK-8D
Ultraviolet-visible spectrophotometer	Thermo	NanoDrop ONE
			Capillary electrophoresis apparatus	Sciex	PA800plus

2. Experimental reagent consumable

Table 2 reagent consumable

3. Sample to be measured

TABLE 3 sample to be tested

Sample name/lot number	Initial concentration (mg/mL)	Use of the same
			Recombinant human heavy chain ferritin-1 (HFn-1)	17.789	Examples 1-2 and 4
Recombinant human heavy chain ferritin-2 (HFn-2)	21.50	Examples 4 to 5
			Recombinant human heavy chain ferritin-3 (HFn-3)	14.89	Example 6
IgG control (M807312)	1	Example 5
			BSA(WXBB6634V)	2	Example 5
Fermentation supernatant	About 3.8	Example 3

The preparation method of the recombinant heavy chain ferritin comprises the following steps:

(1) Seed culture: coli BW25113 containing the human heavy chain ferritin subunit coding sequence was thawed at room temperature, 1mL of seed was inoculated into a conical flask (500 mL) containing 40mg/L of streptomycin sulfate in LB medium at 0.5% (v/v) to obtain a seed liquid, and when the OD600 of the seed liquid was 1.6, the seed liquid was inoculated into a fermenter containing a fermentation medium by flame inoculation at 0.5% of the inoculum size.

The fermentation medium consists of the following components: 14g/L yeast peptone, 10g/L yeast extract, 0.5g/L sodium chloride, 0.5g/L ammonium chloride, 10g/L disodium hydrogen phosphate dodecahydrate, 1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate and 12g/L glucose.

(2) Fermentation culture:

Maintaining the dissolved oxygen concentration (DO) of the fermentation medium at 23%, fermenting at pH 6.5 and temperature of 35deg.C; when the OD600 is 45, 2g/L of arabinose is added to start the expression of the target protein, and fermentation is stopped after 10 hours of induction, so that fermentation broth is obtained. The method for detecting the carbon source in the fermentation liquid comprises the steps of sampling fermentation samples every 4 hours, and detecting OD600, pH and residual sugar.

(3) And (3) thallus collection: centrifuging 20ml fermentation liquor at 2 ℃ at 5000rpm for 15min, and collecting thalli;

(4) Sample preparation: taking the collected thalli, re-suspending the thalli to 20mL by using 15mM Tris-HCl (pH 8.0) buffer solution, cracking the thalli for 2 times by using a high-pressure homogenizer at 900bar, centrifuging the thalli at 8000rpm for 15min, and taking supernatant to obtain fermentation supernatant;

(5) Protein purification: filtering the fermentation supernatant obtained in the step (4), capturing by a Q XL gel column (GE company), purifying by Phenyl Sepharose HP (GE company), desalting, changing the liquid, and preserving at-80 ℃ to obtain the recombinant human heavy chain ferritin. Stored for later use in experiments.

Three batches of HFn samples, designated HFn-1, HFn-2 and HFn-3 (shown in Table 3), were obtained in the above preparation method, and used in the various examples.

EXAMPLE 1 effect of TFA pretreatment protein on SDS-PAGE analysis

This example was pretreated with TFA (room temperature) instead of human heavy chain ferritin of group Wen Duichong, and then analysed by SDS-PAGE.

This example prepared 2 test pieces: 0.1% TFA treatment of the sample and high temperature treatment of the sample at 95 ℃. Table 4 gives the test article preparation schemes.

Table 4 test sample preparation protocol

Results and analysis:

SDS-PAGE analysis was performed on the 0.1% TFA treated sample and the 95℃high temperature treated sample, respectively, using a 12% SDS-PAGE gel, and the results are shown in FIG. 1. FIG. 1 shows that after treatment of ferritin with TFA at room temperature, the small molecular weight protein bands are significantly reduced relative to high temperature (95-10 min), so that the method of pretreatment of ferritin with TFA (room temperature) has higher purity analysis accuracy than the high temperature treatment.

EXAMPLE 2 TFA Effect of pretreatment proteins on CE-SDS analysis

This example was pre-treated with TFA (room temperature) instead of human heavy chain ferritin from group Wen Duichong, and then subjected to CE-SDS analysis.

CE-SDS general parameters:

Detection wavelength: UV 220nm (bandwidth 10 nm) sampling frequency: 2Hz

Filtering mode: normal sample chamber temperature: capillary temperature at 25 ℃): 25 DEG C

CE-SDS running parameters:

Pretreatment of the capillary: the 0.1mol/L sodium hydroxide solution was rinsed at 70psi for 3 minutes, then 0.lmol/L hydrochloric acid solution was rinsed at 70psi for 1 minute, and finally pure water was rinsed at 70psi for 1 minute.

Prefilling of the capillary: SDS gel separation buffer was washed at 70psi pressure for 10 minutes.

Sample injection: 5KV reversed-phase polarity electric sample injection is carried out for 20 seconds.

Sample separation: run at 15KV for 30min with reversed polarity.

A variety of test pieces were prepared in this example. Table 5 gives the test article preparation schemes.

Table 5 test sample preparation protocol

The results of the CE-SDS assay are shown in Table 6.

TABLE 6 influence of different pretreatment methods on CE-SDS results of ferritin (HFn)

Treatment method	MW < HFn subunit	HFn subunit	MW > HFn subunit
				HFn-60min	6.735	80.917	12.348
HFn-95-5min	3.175	92.542	4.283
				HFn-0.1％TFA-5min	1.986	93.545	4.469
HFn-0.1％TFA-30min	1.955	93.676	4.369
				HFn-0.1％TFA-60min	1.905	93.542	4.553
HFn-0.5％TFA-5min	1.721	93.842	4.437
				HFn-0.5％TFA-30min	1.708	93.941	4.351
HFn-0.5％TFA-60min	1.656	94.007	4.337
				HFn-1％TFA-5min	1.608	93.905	4.487
HFn-1％TFA-30min	1.675	93.851	4.474
				HFn-1％TFA-60min	1.675	93.919	4.406
HFn-0.1％TFA-10％ACN-5min	2.102	93.441	4.457
				HFn-0.1％TFA-10％ACN-30min	2.045	93.555	4.4
HFn-0.1％TFA-10％ACN-60min	1.955	93.508	4.537
				HFn-0.1％TFA-15％ACN-5min	1.922	93.677	4.401
HFn-0.1％TFA-15％ACN-30min	1.913	93.794	4.293
				HFn-0.1％TFA-15％ACN-60min	1.824	93.573	4.603
HFn-0.1％TFA-25％ACN-5min	2.133	94.032	3.835
				HFn-0.1％TFA-25％ACN-30min	2.08	93.519	4.401
HFn-0.1％TFA-25％ACN-60min	1.94	93.74	4.32
				HFn-10％ACN-5min	14.852	71.948	13.2
HFn-10％ACN-30min	17.836	67.256	14.908
				HFn-10％ACN-60min	17.273	69.517	13.21
HFn-30％ACN-5min	5.989	85.674	8.337
				HFn-30％ACN-30min	10.222	81.401	8.377
HFn-30％ACN-60min	7.84	81.297	10.863

In table 6, the column under "MW < HFn subunit" represents the percentage of protein peak area for components having a molecular weight less than HFn subunit; the column "MW > HFn subunit" indicates the percentage of protein peak area for components having a molecular weight greater than HFn subunit. The data in Table 6 shows that the proportion of small molecular weight hybrid protein (MW < HFn subunits) of HFn treated at 95℃for 5 minutes is significantly higher than that of the samples treated with TFA treatment system (3.175% vs 1.607-1.986%), whereas the proportion of HFn subunits obtained by TFA pretreatment is significantly higher than that of the samples pretreated at high temperature (94.007vs 92.542), indicating that the small molecular weight hybrid protein content of the protein obtained using the pretreatment method of the invention is lower and that the ferritin subunits are depolymerized more thoroughly and completely. In addition, under the conditions of different concentrations of TFA (0.1% -1%) and different treatment times (5-60 min), the purity of the protein sample is very stable, and the protein sample cannot be obviously changed along with the change of the concentration of TFA and the extension of the treatment time.

Fig. 2-9 show the off-peak behavior of ferritin under different conditions. Wherein:

Figure 2 shows a superposition of 3 Blank control (Blank) systems of recombinant human heavy chain ferritin, showing that neither TFA nor acetonitrile in 3 Blank systems produced interfering peaks.

FIG. 3 shows a CE-SDS overlay of recombinant human heavy chain ferritin in 0.1% TFA system and 95℃high temperature system. Fig. 4 is a partial enlarged view of fig. 3. FIGS. 3 and 4 show that ferritin CE-SDS shows a peak similar to the Wen Chufeng peak at 95℃in the presence of TFA.

FIG. 5 shows a CE-SDS overlay of recombinant human heavy chain ferritin treated in 0.1% TFA system for different times. Fig. 6 is a partial enlarged view of fig. 5. FIGS. 5 and 6 show that ferritin shows similar peak behavior when treated with a 0.1% TFA system for different times of CE-SDS. In addition, the results of treatment with CE-SDS at different times with 0.25% TFA and 0.5% TFA system were similar.

FIG. 7 shows CE-SDS overlays of recombinant human heavy chain ferritin treated with 0.1% TFA-10% ACN system at different times. FIG. 7 shows that ferritin shows similar peak behavior when treated with a 0.1% TFA-10% ACN system for different times of CE-SDS. In addition, the results of treatment with CE-SDS at different times under the 0.25% TFA-10% ACN and 0.5% TFA-10% ACN systems are similar.

FIG. 8 shows a superposition of CE-SDS of recombinant human heavy chain ferritin treated in different systems (HFn-60 min, HFn-95-5min, 0.1% TFA-60min and 0.1% TFA-50% ACN-60 min). Fig. 9 is an enlarged view of a portion of fig. 8. FIGS. 8 and 9 show that ferritin shows similar peak behavior in CE-SDS at different systems.

FIG. 10 shows a CE-SDS overlay of recombinant human heavy chain ferritin treated at different ACN concentration systems for different times. Fig. 11 is a partial enlarged view of fig. 10. Fig. 10 and 11 show that the treatment system with acetonitrile alone is not capable of depolymerizing ferritin subunits (Fn sub).

From table 6 and fig. 2-11 the following conclusions can be drawn:

1) The same sample is subjected to different pretreatment modes, the TFA pretreatment system can depolymerize ferritin subunits more thoroughly and completely, the CE-SDS purity detection result is about 1.4% higher than that of the sample which is most obviously treated at the temperature of 95 ℃ for 5 minutes, and the purity of the sample is very stable under the TFA treatment system and cannot be reduced along with the extension of the TFA acid treatment time;

2) Acetonitrile does not depolymerize ferritin subunits and cannot be used as a pretreatment reagent, but acetonitrile is a common reagent for subsequent protein HPLC analysis;

3) Acetonitrile is added to the TFA treatment system, so that the depolymerization of ferritin subunits is not affected, and the purity result is consistent with that of a pure TFA treatment system, so that acetonitrile can be added to the pretreatment system if HPLC analysis is needed for protein later.

EXAMPLE 3 effect of TFA pretreatment of protein fermentation broth on SDS-PAGE analysis

In this example, TFA (room temperature) was used instead of human heavy chain ferritin fermentation supernatants from group Wen Duichong for pretreatment and SDS-PAGE analysis was performed. This example prepared 2 test pieces: 0.1% TFA treated fermentation supernatant (TFA) and 95-10 min treated fermentation supernatant (95 ℃).

Table 7 gives the test article preparation schemes.

TABLE 7 test sample preparation protocol

Results and analysis:

the SDS-PAGE analysis protocol for the samples is given in Table 8.

TABLE 8 SDS-PAGE analysis protocol for samples

Lanes	Sample name	Sample loading amount
			1	Fermentation supernatant (95 ℃ C.)	10μL
2	Fermentation supernatant (TFA)	10μL

FIG. 12 shows the SDS-PAGE detection of recombinant human heavy chain ferritin fermentation supernatants. The results in FIG. 12 show that the SDS-PAGE results are similar after the same fermentation supernatant is treated with high temperature (95-10 min) and TFA (0.1% final concentration), indicating that it is still effective for pretreatment of unpurified fermentation supernatant with TFA instead of high temperature.

Example 4 TFA applicable interval of TFA concentration in treatment System

This example investigated the effect of different concentrations of TFA and addition of reducing agent on the treatment effect.

The samples were divided into the following 2 groups according to whether the loading buffer contained a reducing agent:

Packet 1: using a reducing loading buffer solution containing a reducing agent;

group 2: a non-reducing loading buffer was used, without reducing agent.

Table 9 gives the test article preparation protocol designs.

TABLE 9 design of test sample preparation protocol

Table 10 test sample preparation protocol

Results and analysis:

The SDS-PAGE analysis protocol for the samples using the non-reducing loading buffer is shown in Table 11. FIG. 13 shows the result of SDS-PAGE (12% SDS-PAGE gel) after HFn treatment with non-reducing loading buffer.

TABLE 11 SDS-PAGE analysis protocol for samples

The SDS-PAGE analysis protocol for the samples using the reduction loading buffer is shown in Table 12. FIG. 14 shows the SDS-PAGE (12% SDS-PAGE gel) results of recombinant human heavy chain ferritin treated with a reduction loading buffer.

TABLE 12 SDS-PAGE analysis protocol for samples

The gel results in FIGS. 13 and 14 show that the ferritin samples can obtain better SDS-PAGE patterns under the conditions of non-reduction and reduction at the concentration of TFA treatment from low to high in the set 4 groups, so that the ferritin samples can have better pretreatment effect in the range of 0.05% -0.5% TFA no matter whether reducing agent exists or not.

Example 5 Effect analysis of TFA System on different protein samples

In this example, different types of protein samples (IgG control, BSA, ferritin) were treated separately with TFA system pair and analyzed by SDS-PAGE.

Table 13 gives the test article preparation protocol designs.

TABLE 13 design of test sample preparation protocol

Table 14 gives the test article preparation schemes.

Table 14 test sample preparation protocol

Results and analysis:

The SDS-PAGE analysis protocol for the samples is given in Table 15.

TABLE 15 SDS-PAGE analysis protocol for samples

FIG. 15 shows that samples treated with ferritin in both reduced and non-reduced loading buffers were tested consistently, but that treatment at 95℃produced more small molecule impurities than TFA treatment.

The SDS-PAGE analysis protocol for the samples is given in Table 16.

TABLE 16 SDS-PAGE analysis protocol for samples

FIG. 16 shows the SDS-PAGE (10% SDS-PAGE gel) results of BSA and IgG.

FIG. 16 shows that the detection results of BSA treated with TFA and 95℃are similar, but that the amount of fragments after 95℃treatment is significantly greater, whether under non-reducing or reducing treatment conditions, indicating that the TFA system can be used instead of high temperature for SDS-PAGE sample treatment of BSA.

In addition, FIG. 16 shows that the electrophoresis effect of IgG after TCEP reduction treatment at normal temperature of TFA (lane 9) is substantially the same as that of 100 ℃ C./95 ℃ C. Reduction treatment at high temperature, which indicates that the pretreatment method of TFA can be used instead of the high temperature treatment method in the presence of a reducing agent.

EXAMPLE 6 Effect of pre-addition of SDS to protein TFA treatment System on CE-SDS analysis

This example shows the effect of SDS pre-addition to ferritin TFA treatment system on the CE-SDS assay

Table 17 gives the test article preparation schemes.

Table 17 test article preparation protocol

Results and analysis:

FIG. 17 shows a superimposed graph of CE-SDS detection of samples after treatment of ferritin with TFA and SDS at various concentrations for various times. Fig. 18 is a partial enlarged view of fig. 17.

Table 18 gives the data statistics.

Table 18 data statistics table

Treatment method	MW < HFn subunit	HFn subunit	MW > HFn subunit
				HFn-0.5％TFA-30min	1.791	93.841	4.368
HFn-0.25% SDS-0.5% TFA-immediate detection	1.303	94.543	4.154
				HFn-2.5％SDS-0.5％TFA-30min	2.026	93.645	4.329
HFn-2.5％SDS-0.5％TFA-60min	2.204	93.71	4.086
				HFn-5% SDS-0.5% TFA-immediate detection	1.428	94.392	4.18
HFn-5％SDS-0.5％TFA-30min	2.052	93.668	4.28
				HFn-5％SDS-0.5％TFA-60min	2.003	93.719	4.278

FIGS. 17, 18 and statistics Table 18 show that after ferritin is treated by TFA system containing different concentrations of SDS, the peak-to-peak pattern of CE-SDS remains consistent, while the data table shows that increasing the concentration of SDS in the treatment system for 30-60 minutes does not increase the main peak percentage (i.e., the peak of the protein of interest).

In addition, the data in the data statistics table also show that the purity of the sample to be tested with the same protein concentration prepared by the same system is higher than that of the sample to be tested after being treated for 30-60 minutes, the purity of the sample after being placed in a sample chamber at 10 ℃ for a period of time is about 7%, and the main peak area of the sample to be immediately sampled is about 50%. The possible reasons for this are: 1) Prolonged periods of acid conditions may lead to increased ferritin hydrolysis and thus reduced purity; 2) TFA concentration decreases after addition of sample buffer, acidity decreases, and long standing times may result in a re-polymerization of part of ferritin, thus greatly decreasing the main peak area. 3) The temperature of the sample system after being placed in the 10 ℃ sample chamber for a period of time is lower than the temperature (room temperature) of the sample detected by immediate sample introduction, so that the viscosity is higher, and the difference between the two sample introduction is caused.

Therefore, the addition of SDS in the TFA system has no influence on the subsequent detection analysis, and for CE-SDS detection, the sample injection detection effect is better immediately after the sample preparation.

Claims

1. An identification assay method for a purified protein, the method comprising:

(1) Pretreatment of purified protein: pretreating the purified protein with a low concentration of trifluoroacetic acid to obtain a pretreated purified protein, wherein the working concentration of the low concentration of trifluoroacetic acid is 0.5% -1% v/v;

(2) And (3) identifying and detecting the purified protein: identifying the purity of the pretreated purified protein obtained in the detection step (1) by adopting sodium dodecyl sulfate capillary electrophoresis or sodium dodecyl sulfate polyacrylamide gel electrophoresis,

The purified protein is contacted with trifluoroacetic acid in a manner selected from the group consisting of: adding the purified protein to a solution containing trifluoroacetic acid, adding the purified protein and trifluoroacetic acid to the solution simultaneously, or adding trifluoroacetic acid to the solution of purified protein; mixing the purified protein with trifluoroacetic acid;

after the purified protein is mixed with trifluoroacetic acid, the mixture is placed for more than 5 minutes at the temperature of 4-25 ℃;

the purified protein is recombinant human heavy chain ferritin.

2. The method of claim 1, wherein the time of placement is 5-60 minutes.

3. The method of claim 2, wherein the time of placement is 5-30 minutes.

4. The method according to claim 1, wherein the purified protein is obtained by subjecting the protein to a purification treatment by a molecular biological method and by subjecting the protein to an induction expression.

5. The process of claim 1, wherein the low concentration trifluoroacetic acid has a working concentration of 0.5% v/v.

6. The method of any one of claims 1-5, wherein the concentration of protein in the purified protein ranges from 0.2 to 20 mg/ml.

7. The method of claim 6, wherein the concentration of protein in the purified protein is in the range of 0.2-10mg/ml.

8. The method of claim 7, wherein the concentration of protein in the purified protein is in the range of 0.5-5 mg/ml.

9. The method of claim 8, wherein the concentration of protein in the purified protein is in the range of 0.75-5 mg/ml.

10. The method of claim 1, wherein a protein reducing agent is added to the method.

11. The method of claim 10, wherein the reducing agent is selected from one or more of beta-mercaptoethanol, dithiothreitol, tris (2-carboxyethyl) phosphine, or SDS.

12. The method of claim 11, wherein the reducing agent is added to the solution system prior to, simultaneously with, or after contacting the protein with trifluoroacetic acid.

13. The method of claim 11, wherein the concentration of beta-mercaptoethanol is 2-5% v/v.

14. The method of claim 11, wherein the dithiothreitol concentration is 50-100 mM.

15. The method of claim 11, wherein the tris (2-carboxyethyl) phosphine has a concentration of 5-50 mM.

16. The method of claim 11, wherein the concentration of SDS is 1-3% by mass/volume.

17. The process according to any one of claims 1 to 5, wherein acetonitrile is added to the low concentration trifluoroacetic acid, wherein the working concentration of acetonitrile in the pretreatment system is 5% to 60% v/v and the working concentration of trifluoroacetic acid is 0.5% to 1% v/v.

18. The process of claim 17, wherein the working concentration of acetonitrile is 10% -50% v/v.

19. The process of claim 18, wherein the working concentration of acetonitrile is 15% -30% v/v.

20. The method of claim 17, wherein the working concentration of trifluoroacetic acid is 0.5% v/v.

21. The method of claim 1, wherein the solution is an aqueous solution.

22. The method of claim 21, wherein the solution is a physiological buffer.

23. Use of the method for the identification and detection of a purified protein according to any one of claims 1 to 22 in the detection of protein purity.