CN118221796A

CN118221796A - Method for purifying tissue factor and buffer composition for purification thereof

Info

Publication number: CN118221796A
Application number: CN202311829237.6A
Authority: CN
Inventors: 严俊; 蒋友红; 卿小红
Original assignee: Baorui Biotechnology Taizhou Co ltd
Current assignee: Baorui Biotechnology Taizhou Co ltd
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-06-21

Abstract

The tissue factor purifying method and the composition for purifying are provided, and trehalose is added in the whole purifying process to improve the stability of the tissue factor in a solution, effectively avoid the formation of precipitation and improve the yield of the tissue factor.

Description

Method for purifying tissue factor and buffer composition for purification thereof

Technical Field

The present invention relates to the field of assays, and in particular to methods for purifying tissue factor for mammals, and buffer compositions for use in purification.

Background

The mammalian tissue factor is a transmembrane glycoprotein with a molecular weight of about 42kDa. The coagulation activation pathway is initiated after formation of a complex with the coagulation factor FVII or FVIIa. Many types of cells express tissue factors, but under normal conditions, blood does not contact tissue factors on the surface of these cells due to the barrier action of blood vessels; when the integrity of the blood vessel is destroyed due to the tissue injury, the tissue factor is exposed at the damaged blood vessel, and the coagulation cascade is activated accordingly.

Thrombin is mostly derived from the leachate of rabbit brain, placenta and lung tissue. Thrombin of PT reagent of sysmex is derived from placenta, PT thromboplastin of stago and Shanghai sun is derived from rabbit brain tissue. Because of different sources, different concentrations and different sensitivities of the thromboplastin of different companies, the problems of poor product quality, instability, poor sensitivity and the like are caused. The major components of thromboplastin are Tissue Factor (TF) and phospholipids.

Tissue Factor (TF) is a transmembrane single-chain glycoprotein consisting of 260 amino acid residues and having a molecular weight of about 47kDa. The membrane-spanning region has three structural domains, an extracellular region, a transmembrane region and an intracellular region, and mainly plays roles in that the extracellular region contains 3-5 disulfide bond structures, has serine protease activity, is an important site for activating blood coagulation, and immediately after 23 amino acids penetrate through a cell membrane, so that the membrane-spanning region is combined with phospholipid on the surface of the cell membrane, and a surface combined with downstream factors including Ca2+ and VII (seven factors) is provided.

Disclosure of Invention

The recombinant expressed transmembrane protein is easy to form precipitate in the purification process to lose protein activity, so the invention provides a purification method of tissue factor and a buffer composition for purification.

In the purification process of the tissue factor, HEPES is used for preparing the buffer solution, the pH value of the buffer solution is less influenced by the environmental temperature, and the buffer solution is more suitable for purifying the tissue factor, so the HEPES is used in all the buffer solutions. 2. Selecting 5mM imidazole from a bacteria breaking buffer solution and a loading buffer solution, wherein the recovery rate is high and the target protein column hanging rate is high; 3. the concentration of imidazole is selected from the balance buffer solution and the elution buffer solution, and the concentration of trehalose is selected to be 5%, so that the activity of target protein can be obviously reserved, the stability of tissue factor in the solution is improved, the formation of sediment is effectively avoided, and the yield of the tissue factor is improved.

The molecular structure of the key raw material tissue factor used in the invention comprises 3 structural domains: extracellular domain, transmembrane domain, cytoplasmic domain. The amino acid sequence of the tissue factor is shown as SEQ No. 1, and the SEQ No. 1 has the following sequence:

adttgray nltwkstnfk tilewepksi dhvytvqist rlenwkskcf ltaetecdlt devvkdvgqt

ymarvlsypa rngnttgfpe eppfrnspef tpyldtnlgq ptiqsfeqvg tklnvtvqda rtlvrrngtf

lslravfgkd lnytlyywra sstgkktatt ntneflidvd kgenycfsvq avipsrkrkq rspesltect

sreqgrarem ffiigavvvv alliivlsvt vykcrkarag psgkesspln ia；

Based on the special structure of the tissue factor, the tissue factor is easy to form precipitate due to being separated from the original reducing environment in the purification process, and the recovery rate is low. Therefore, the invention provides a tissue factor purification method, trehalose is added in the whole purification process to improve the stability of the tissue factor in the solution, effectively avoid the formation of sediment and improve the yield of the tissue factor.

Compared with the prior art, the invention has the beneficial effects that:

1. in the purification process of the tissue factor, HEPES is used for preparing a buffer solution, the pH value of the buffer solution is less influenced by the environmental temperature, and the buffer solution is more suitable for purifying the tissue factor, so the HEPES is used in all the used buffer solutions;

2. Selecting 5mM imidazole from a bacteria breaking buffer solution and a loading buffer solution, wherein the recovery rate is high and the target protein column hanging rate is high;

3. The proper imidazole concentration is selected from the balance buffer solution and the elution buffer solution, and the trehalose concentration of 5% is selected, so that the activity of the target protein can be obviously reserved, the stability of the tissue factor in the solution is improved, the formation of sediment is effectively avoided, and the yield of the tissue factor is improved.

Abbreviations

IPTG isopropyl-beta-D thiogalactoside

CHAPS 3- [ (3-cholamidopropyl) dimethylamino ] propane sulfonic acid inner salt

HEPES 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid

RTF protein recombinant tissue factor protein

BL21DE3 host bacterium

Drawings

FIG. 1 shows an electrophoretic test of 3 different imidazole concentrations in equilibration buffer versus hetero-and target proteins;

FIG. 2 shows an electrophoretic test of 3 different imidazole concentrations in elution buffer versus hetero-and target proteins;

FIG. 3 is a graph of observed precipitation of purification during tissue factor purification using different concentrations of trehalose (2%, 5% and 10%);

Detailed Description

The invention is further illustrated by the following examples

EXAMPLE 1 construction and screening of engineered cells

Sequence source: NCBI Access, AAA63469.1;

And (3) construction: designing nucleic acid sequence and synthesizing, introducing into recombinant plasmid, transforming and introducing into expression cell competence to obtain recombinant strain.

Expression vector/host: pET32a (+)/BL 21DE3.

The specific method comprises the following steps: the rabbit tissue factor gene sequence is from Genbank, NCBI Reference Sequence:AAA63469.1, the full-length sequence is selected, the sequence is synthesized by Shanghai biological company through designing primer, cloned into pET32a (+) plasmid through enzyme cutting enzyme connection, and transformed into host bacterium BL21DE3 for subsequent strain fermentation and protein purification.

Example 2. Amplification and fermentation methods of the species were as follows:

a transformed glycerol strain of example 1 was removed from the-80℃refrigerator and inoculated in LB medium (50. Mu.g/mL ampicillin) in an ultra-clean bench. Culturing at 37deg.C and 200rpm overnight in a shaker for 12-16h. The next day, the flask was turned to a flask according to the bacterial liquid and medium 1:25, and ampicillin was added to a final concentration of 50. Mu.g/mL. After further culturing for 3 hours, detecting OD ₆₀₀ value of 0.6-1.0 by using an ultraviolet spectrophotometer, performing IPTG (isopropyl-beta-D thiogalactoside) induction, expressing protein at a low temperature of 16 ℃ until the final concentration of IPTG is 1mM, and collecting the bacterial cells in the morning on the third day. And (5) centrifuging. The centrifugal bottle is weighed in advance, bacterial liquid is poured into the centrifugal bottle, balancing and centrifuging are carried out, 8000g centrifuging is carried out for 5min, the supernatant is discarded, and weighing is carried out again. The two quality differences are the weight of the wet bacteria.

EXAMPLE 3 screening experiments for purification of buffers used

3.1 Screening of buffer System

	Tris-HCL	HEPES
			Whether or not the pH is affected by temperature	Wide pH variation range	The pH change range is small
Whether or not it is suitable for purification	Is not suitable for	Is suitable for
			Placing the bacterial-destroying supernatant at room temperature for 2 hours	Precipitation occurs	No precipitate

As shown in the table above, when the technical scheme is implemented, the pH value of the HEPES preparation buffer solution is less influenced by the environmental temperature, and the bacterial-destroying supernatant cannot be precipitated. HEPES was shown to be more advantageous for the stability of the target protein, and was suitable for purification of the recombinant tissue factor, and therefore HEPES was selected for use as a buffer.

3.2 Screening of imidazole concentration in the breaking buffer and the Loading buffer

Group 1: bacteria breaking buffer = loading buffer: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

group 2: bacteria breaking buffer = loading buffer: 20mM HEPES (pH 8.0), 150mM NaCl,25mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

	Group 1	Group 2
			Recovery rate	38％	2％
Target protein column hanging rate	99％	10％

As shown in the above table, group 1 containing 5mM imidazole was high in recovery rate and the target protein was high in column yield, so that 5mM imidazole was selected.

3.3 Screening of imidazole concentration in equilibration buffer

Group 1: equilibration buffer: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

Group 2: equilibration buffer: 20mM HEPES,150mM NaCl,20mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

group 3: equilibration buffer: 20mM HEPES,150mM NaCl,25mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

As shown in the above table and FIG. 1, from left to right in FIG. 1, when the first lane is 5mM imidazole in the equilibrium solution, the eluent contains a certain amount of hetero protein and no target protein is eluted; the second lane shows that when the balance liquid contains 20mM imidazole, the content of the hetero protein in the eluent is higher and no target protein is eluted; the third lane shows that when 25mM imidazole is contained in the balance liquid, the impurity protein content in the eluent is higher but a small amount of target protein is eluted; the third lane is Marker. The effect of the binding equilibration buffer is to remove the contaminating proteins, so that the equilibration solution is eventually selected to contain 20mM imidazole.

3.4 Screening of imidazole concentration in elution buffer

Group 1: elution buffer: 20mM HEPES,150mM NaCl,100mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

group 2: elution buffer: 20mM HEPES,150mM NaCl,250mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

group 3: elution buffer: 20mM HEPES,150mM NaCl,500mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

As shown in the table and FIG. 2, from left to right in FIG. 2, the first lane is Marker, the second lane is eluted with 500mM imidazole, a large amount of target protein is eluted, and the content of impurity protein is high; the third lane was eluted with 250mM imidazole, a large amount of the target protein was eluted, almost no contaminating proteins; the fourth lane was eluted with 100mM imidazole, and only a small amount of the target protein was eluted. The purpose of the elution buffer is to finally elute the target product, so 250mM imidazole is chosen as the composition of the elution buffer.

3.5 Trehalose is a non-reducing disaccharide, the effective protective effect on biomolecules is related to its larger hydration volume, the exclusion effect of trehalose in a solution of biomolecules is more remarkable, and the hydrogen bonding ability between trehalose and proteins is stronger than other substances, so trehalose is selected as a stabilizer for experiments.

For the concentration of trehalose, screening experiments were performed, as shown in fig. 3, using different concentrations of trehalose (2%, 5% and 10%) during tissue factor purification, the eluted fractions containing the target protein were broken by 7d at 37 ℃ in an accelerated manner, from left to right, I represents a 2% trehalose group, and as a result, a large amount of protein precipitation occurred; II represents 5% trehalose group, resulting in almost no protein precipitation; III represents 10% trehalose group, resulting in almost no protein precipitation; group II and group III continued to stand for 21d, and no precipitation was found yet. Continued standing for 28d found that precipitation occurred in both groups with no significant difference in the amount of precipitation. Thus, 5% trehalose was chosen as the optimal use concentration.

Example 4: the preferred method determined after the above screening experiments is as follows:

Method 1

1.1, Bacteria-destroying buffer solution: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,

5% Trehalose, ph8.0;

1.2 loading buffer: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,

5% Trehalose, ph8.0;

1.3 equilibration buffer: 20mM HEPES,150mM NaCl,20mM imidazole, 10mM CHAPS,

5% Trehalose, ph8.0;

1.4 elution buffer: 20mM HEPES,150mM NaCl,250mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

Method 2 comparative example

1.1, Bacteria-destroying buffer solution: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS, pH8.0;

1.2 loading buffer: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS, pH8.0;

1.3 equilibration buffer: 20mM HEPES,150mM NaCl,20mM imidazole, 10mM CHAPS, pH8.0;

1.4 elution buffer: 20mM HEPES,150mM NaCl,250mM imidazole, 10mM CHAPS, pH8.0;

4.1 protein purification method is as follows:

4.1.1. the wet bacteria amplified and fermented in example 2 were resuspended in 30mL of "lysis buffer" per gram of wet bacteria and broken three times with a 750bar high pressure ice bath. Centrifuging 12000g of the bacterial cells after bacterial breaking for 20min, and harvesting the supernatant;

4.1.2. And (3) filtering a bacterial-destroying supernatant: filtration was performed using a stainless steel filter (0.22 uM membrane), and the filtrate was collected;

4.1.3. And (3) purifying by using a pre-loaded nickel column, wherein the purification flow rate is kept at 5mL/min in each step, the nickel column is balanced by using 5 column volumes of 'loading buffer', then is loaded, 10 column volumes of 'balancing buffer' are used for balancing after loading, and finally, the sample is eluted by using 3 column volumes of 'eluting buffer', and the eluted sample A is collected. Ultrafiltration concentration, protein concentration was tested with BCA kit.

The purification results obtained are as follows:

As can be seen from the above table, when the method 1 of the present invention is used, the total protein amount in the "bacterial-destroying supernatant" obtained in the step 4.1.2 is 15.8mg, and the "soluble protein yield" is 100%, which are all significantly better than the method 2; likewise, when the method 1 of the present invention was used, the "total protein amount" of the eluted sample obtained after the "nickel column purification" obtained in the step 4.1.3 was 6.4mg, and the "soluble protein yield" was 40.5%, which were significantly superior to the method 2.

4.2 Recombinant tissue factor lipidation:

Tissue factor is a membrane protein, which functions by re-lipidation.

The specific lipidation method is as follows:

The eluted sample A obtained after "nickel column purification" obtained in the 4.1.3. Step was diluted with a equilibration solution to a concentration of 100. Mu.g/mL.

Rabbit cephalin is prepared into 10mg/mL working solution by using a solution (150 mM sodium chloride, 10mM deoxycholate sodium) and is subjected to ultrasonic dissolution.

Lipidation conditions: 50. Mu.L of 100. Mu.g/mL rTF, 50. Mu.L of 10mg/mL rabbit cephalin and 900. Mu.L of HBS buffer (20 mM HEPES,150mM sodium chloride, pH 7.4). The cells were incubated at 37℃for 2h to allow the cells to be lipidated, and the cells were exchanged with buffer 20mM HEPES,150mM sodium chloride, pH7.4, and dialyzed, 3 times at 3-hour intervals. After the dialysis is completed, the lipidation solution is taken out.

Lipid detection: sample 1 and sample 2 are whole blood, derived from two individuals. Plasma was detected on a thromboelastography device. Each test: 1. Mu.L of lipid + 20. Mu.L of 0.2M calcium chloride + 340. Mu.L of plasma.

The activity test results were as follows:

As can be seen from the above table, in sample 1, the TEG ACT (sec) mean value of the method 1 according to the present invention was 113, and the TEG ACT (sec) mean value of the comparative example method 2 was 276.67, which revealed that the TEG ACT value of the method 1 was significantly smaller than that of the method 2, indicating that the activation efficiency of the lipidic compound in the method 1 was high, i.e., the activity of the tissue factor purified by the reaction was high. The above results can also be seen in sample 2.

In summary, the HEPES buffer system is selected and used by respectively screening and optimizing the bacteria breaking buffer, the loading buffer, the balance buffer and the elution buffer; the proper imidazole concentration is selected from different buffer liquid systems, and a specific amount of trehalose is added, so that the stability of the protein is obviously improved, precipitation is not easy to form, and the activity of tissue factor is maximally reserved. The method is simple and easy to operate, has low cost, and provides a simple and effective method for obtaining the high-purity active target protein in the tissue factor purification preparation process.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims

1. A composition for purifying tissue factor, the composition comprising the following components:

Bacteria-destroying buffer solution: HEPES, naCl, imidazole, CHAPS, trehalose;

loading buffer solution: HEPES, naCl, imidazole, CHAPS, trehalose;

Equilibration buffer: HEPES, naCl, imidazole, CHAPS, trehalose; and

Elution buffer: HEPES, naCl, imidazole, CHAPS, trehalose.

2. The composition of claim 1, comprising the following ingredients:

Bacteria-destroying buffer solution: HEPES, naCl,5mM-25mM imidazole, CHAPS,5% trehalose;

loading buffer solution: HEPES, naCl,5mM-25mM imidazole, CHAPS,5% trehalose;

Equilibration buffer: HEPES, naCl,5mM-25mM imidazole, CHAPS,5% trehalose; and

Elution buffer: HEPES, naCl,100-500mM imidazole, CHAPS,5% trehalose.

3. The composition of claim 1, comprising the following ingredients:

bacteria-destroying buffer solution: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

Loading buffer solution: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0;

equilibration buffer: 20mM HEPES,150mM NaCl,20mM imidazole, 10mM CHAPS,5% trehalose, pH8.0; and

Elution buffer: 20mM HEPES,150mM NaCl,250mM imidazole, 10mM CHAPS,5% trehalose, pH8.0.

4. A method of tissue factor purification, the method comprising:

1) Construction and screening of engineering cells: the method comprises the following steps: the rabbit tissue factor gene sequence is from Genbank, NCBI Reference Sequence: AAA63469.1, selecting full-length sequence, synthesizing by designing primer and delivering to Shanghai biological company, cloning to pET32a (+) plasmid by enzyme cutting enzyme connection, and transforming to host bacterium BL21DE3, the strain is used for subsequent fermentation and protein purification;

2) Strain amplification and fermentation: the method comprises the following steps: inoculating the transformed glycerinum in the step 1 to LB culture medium containing 50 mug/mL ampicillin in an ultra clean bench, and culturing overnight; then according to the bacterial liquid and the culture medium 1: adding 25 volume ratio into a large bottle, adding ampicillin, continuously culturing, inducing isopropyl-beta-D thiogalactoside, expressing protein at low temperature of 16 ℃ until overnight, and collecting to obtain thalli;

3) Resuspension of the amplified and fermented wet bacteria with 'bacteria breaking buffer' added to each gram of wet bacteria, high-pressure ice bath crushing, bacteria centrifugation after bacteria breaking, and supernatant collection;

4) Filtering the bacterial-destroying supernatant: filtering with stainless steel filter, and collecting filtrate;

purifying by using a pre-loaded nickel column, balancing the nickel column by using a loading buffer solution, loading a sample, balancing by using the balancing buffer solution after loading, eluting a sample by using an eluting buffer solution, and collecting the eluted sample.

5. A method of purifying a tissue factor according to claim 4, wherein the lysis buffer comprises: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0.

6. A method of purifying a tissue factor according to claim 4, wherein the loading buffer comprises: 20mM HEPES,150mM NaCl,5mM imidazole, 10mM CHAPS,5% trehalose, pH8.0.

7. A method of purifying a tissue factor according to claim 4, wherein the equilibration buffer comprises: 20mM HEPES,150mM NaCl,20mM imidazole, 10mM CHAPS,5% trehalose, pH8.0.

8. The method of tissue factor purification of claim 4, wherein the elution buffer comprises: 20mM HEPES,150mM NaCl,250mM imidazole, 10mM CHAPS,5% trehalose, pH8.0.

9. A tissue factor purification method according to claim 4, wherein the purification flow rate in step 4 is maintained at 5mL/min; the nickel column is balanced by 5 column volumes of loading buffer solution; 10 column volumes "equilibration buffer" were equilibrated and 3 column volumes "elution buffer".

10. Use of a composition according to claim 1 for the preparation of a buffer composition for purifying tissue factor.