RU2434018C2 - Method of obtaining and purifying human inhibin-a - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of obtaining and purifying human inhibin-A. Pieces of placenta are crushed and homogenised. A physiological solution is added in ratio of 1:2. Butyl alcohol is added to the obtained mass in ratio of 1:10 and then left for a day in a refrigerator at t +4°C. The mixture is then centrifuged with diethyl ether in ratio of 1:3 for 10-12 minutes at 5000 rpm in a cold centrifuge twice. Supernatant fluid containing inhibin is put into a flask which is then put into a refrigerator at t +4°C for a day. The supernatant fluid is centrifuged once more and over protein content therein is determined. The obtained solution is mixed with an equal volume of saturated ammonium sulphate solution and centrifuged in a cold centrifuge for 45-50 min at 10000 rpm. The obtained residue is dissolved in a tris-HCl buffer with pH 7.8, and then deposited on a lectin-sepharose column balanced with tris-HCl buffer. The entire volume of the dissolved residue obtained at the previous step is passed through. The column is washed with 1M sodium chloride solution buffered with tris-HCl buffer. The inhibin bound on the column is then eluted with 0.1M lactose solution in a borate buffer at pH 9.0. The eluate undergoes dialysis and concentration.

EFFECT: method enables to obtain inhibin-A with high output, high specific activity and high degree purification.

1 tbl, 3 ex

Description

The invention relates to medicine, namely biochemistry, and can be used to obtain and purify inhibin-A.

From the practice of medicine, a method for isolating and purifying a protein is known (“A method for producing the antimicrobial peptide of arenicin”, application No. 2006121111/13 dated 06.16.2006; patent No. 2316595), which consists in cultivating a recombinant producer strain Escherichia coli BL21 (DE3) / pE-His8 -TrxL-Ar2 in a nutrient medium, destruction of the obtained cells, isolation of inclusion bodies containing the His8-TrxL-Ar2 fusion protein; purification of the hybrid protein by affinity chromatography, solubilization of the protein and cleavage with bromine cyan in an acidic medium, separation of the reaction products, purification of the target product by reverse phase HPLC.

The disadvantage of this method is the low yield of the recombinant peptide of 5 mg per 1 liter of cell culture, the double use of chromatography, which increases the cost of the method.

There is also known a method of obtaining a concentrate of factor IX of the blood coagulation system from the patent "Concentrate of factor IX of the blood coagulation system and method of obtaining it", application No. 20010117508/14 from 05.07.2000; Patent No. 2163140; including: sorption of factor IX from donor plasma products on the DEAE - Toyopearl anion exchanger, washing of the anion exchanger, centrifugation, filtration, sorption on a column with an anion exchanger, elution in a stepwise gradient of ionic strength 200-360 mmol Na ⁺ / l 0.01-0.02 M Tris-HCl buffer solution with a pH of 7.0-7.5 containing 0.05-0.07 M glycine and 0.05-0.06 M trisubstituted sodium citrate, dilution with start buffer (pH 7.2-7.8 ) to a protein concentration of 0.35-0.50%, sterilizing filtration.

The disadvantage of this method is the low yield of the target product and low reproducibility, which leads to the need to increase the cost of obtaining the target product.

There is also a method of producing RNP (low molecular weight ribonucleopeptides) from cell nuclei from the patent “Means that stimulates the repair of damage, possessing tissue, organo- and stage-specificity and antiviral activity”, application No. 2003101855/13 of 24.01.2003, patent No. 2238756; including: washing the liver tissue of cattle from blood cells in physiological saline, homogenizing at low temperature for 5 minutes in a solution containing 0.01 M Tris-HCl buffer pH 8.5, 0.001 M calcium chloride, 0.001 M EDTA and 0 , 6% Triton X-100, centrifugation, isolation of the cytoplasmic RNP fraction, washing, suspension in a solution of sodium acetate containing 0.02 M Tris-HCl buffer pH 5.0, 0.001 M EDTA and 0.5% sodium dodecyl sulfate, pouring the mixture phenol-chloroform (1: 1), heating for 10 minutes in a water bath at a temperature ature 40 ° С, cooling, centrifuging at 5000 rpm for 20 minutes, selecting the upper phase, pouring 350 ml of a phenol-chloroform mixture (1: 1), heating, centrifuging, adding an equal volume of acetate buffer and a phenol-chloroform mixture, heating at a temperature of 50 ° C, cooling, centrifugation, sequentially repeating this procedure at 60, 70 and 80 ° C, deproteinization of the phases of centrifugates, pouring 2.5 volumes of chilled ethanol, separating the precipitate, washing with ethanol, and sterilization by filtration.

The disadvantage of this method is the multi-stage, low degree of reproducibility due to the impossibility of automation, which complicates the process and increases the cost of the target product.

Closest to the claimed invention is a method of "Cleaning and partial characterization of inhibin in porcine follicular fluid" described in the journal "Biochemical and biophysical research communications", Vol.133, No. 1, 1985, November 27, 1985, 120-127, which consists in preparing an extract from biological tissues, sequential precipitation of the protein with organic solvents and ammonium sulfate, by reverse phase HPLC and gel-penetrating LC.

The disadvantages of the prototype are small volumes (in μl and μg), multi-stage, double use of chromatography, low reproducibility, which complicates the process and increases the cost of the method.

The task of the invention is to simplify the process of obtaining and purification of inhibin-A.

The problem is solved by the fact that pieces of the placenta are crushed and homogenized, physiological saline is added in a ratio of 1: 2, butyl alcohol is added to the resulting mass at a rate of 1:10 and left for a day in the refrigerator at t + 4 ° C, then the mixture is centrifuged with ether 1: 3 for 10-12 minutes at 5000 rpm in a refrigerator centrifuge twice, the supernatant containing inhibin is placed in a flask in a refrigerator at t + 4 ° C for a day, then centrifuged again and the total protein in the supernatant is determined the resulting solution with The solution is sedimented with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 45-50 minutes at 10,000 rpm, the resulting precipitate is dissolved in TRIS-HCl buffer with a pH of 7.8, applied to a TRIS-HCl lectin-Sepharose column. with a buffer, the entire volume of the dissolved precipitate obtained in the previous step is passed through, the column is washed with a 1 M solution of sodium chloride buffered with TRIS-HCl buffer, then the inhibin-A bound with a 0.1 M solution of lactose in borate buffer, pH 9.0, is eluted dialyze and concentrate digging.

The proposed method for the preparation and purification of human inhibin-A was successfully tested on 63 samples of human placenta in the practical work of the Department of General and Bioorganic Chemistry of the Astrakhan State Medical Academy during 2009.

The proposed method is as follows.

Primary placenta extract

To prepare the extract, pieces of the placenta were crushed and homogenized by adding saline in a ratio of 1: 2. (Total volume 1200 ml, total protein 31640 mg, inhibin-A 164 mg, 100% yield, purification 1.)

Butanol fraction

To the resulting mass was added butyl alcohol in the calculation of 1:10 and left for a day in the refrigerator (+ 4 ° C). Then the mixture was centrifuged with diethyl ether (1 part ether and 3 parts homogenate) for 10 minutes at 5000 rpm in a refrigerator centrifuge twice. The supernatant (445 ml) containing inhibin was placed in a flask in a refrigerator (+ 4 ° C) for a day. Then centrifuged again and determined the total protein in the supernatant (7680 mg), the amount of inhibin-A 95 mg.

Yield 57.9%, degree of purification 2.3.

Ammonium sulfate precipitation

The resulting solution was mixed with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 45 minutes at 10,000 rpm. The resulting precipitate was dissolved in TRIS-HCl buffer with a pH of 7.8. (Total volume 115 ml, total protein 855 mg, amount of inhibin-A 73 mg, yield 44.5%, purification 16.5.)

Affinity chromatography

This stage of purification of inhibin-A is based on the ability of castor bean lectin (ricin) to precipitate inhibin-A that we discovered. Represents inhibin-A affinity chromatography on immobilized castor bean lectin. Through a column (2 × 14 cm) of lectin-sepharose balanced with TRIS-HCl buffer, the entire volume of the dissolved precipitate obtained in the previous step was passed. Next, the column was washed with a 1 M solution of sodium chloride, buffered with TRIS-HCl buffer, and then the column bound inhibin-A was eluted with a 0.1 M solution of lactose in borate buffer, pH 9.0. The resulting eluate was dialyzed and concentrated to a volume of 13 ml (total protein 32 mg, amount of inhibin-A 27 mg, yield 16.3%, purification 138.8).

Examples using different esters

Example 1

Primary placenta extract

To prepare the extract, pieces of the placenta were crushed and homogenized by adding saline in a ratio of 1: 2. (Total volume 1200 ml, total protein 31640 mg, inhibin-A 164 mg, 100% yield, purification 1.)

Butanol fraction

To the resulting mass was added butyl alcohol in the calculation of 1:10 and left for a day in the refrigerator (+ 4 ° C). The mixture was then centrifuged with dipropyl ether (1 part ether and 3 parts homogenate) for 10 minutes at 5000 rpm in a refrigerator centrifuge twice. The supernatant (445 ml) containing inhibin was placed in a flask in a refrigerator (+ 4 ° C) for a day. Then centrifuged again and determined the total protein in the supernatant (7680 mg), the amount of inhibin-A 95 mg.

Yield 57.9%, degree of purification 2.3.

Ammonium sulfate precipitation

The resulting solution was mixed with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 45 minutes at 10,000 rpm. The resulting precipitate was dissolved in TRIS-HCl buffer with a pH of 7.8. (Total volume 115 ml, total protein 855 mg, amount of inhibin-A 73 mg, yield 44.5%, purification 16.5.)

Affinity chromatography

This stage of purification of inhibin-A is based on the ability of castor bean lectin (ricin) to precipitate inhibin-A that we discovered. Represents inhibin-A affinity chromatography on immobilized castor bean lectin. Through a column (2 × 14 cm) of lectin-sepharose balanced with TRIS-HCl buffer, the entire volume of the dissolved precipitate obtained in the previous step was passed. Next, the column was washed with a 1 M solution of sodium chloride, buffered with TRIS-HCl buffer, and then the column bound inhibin-A was eluted with a 0.1 M solution of lactose in borate buffer, pH 9.0. The resulting eluate was dialyzed and concentrated to a volume of 13 ml (total protein 32 mg, amount of inhibin-A 27 mg, yield 16.3%, purification 138.8).

Example 2

Primary placenta extract

To prepare the extract, pieces of the placenta were crushed and homogenized by adding saline in a ratio of 1: 2. (Total volume 1200 ml, total protein 31640 mg, inhibin-A 164 mg, 100% yield, purification 1.)

Butanol fraction

To the resulting mass was added butyl alcohol in the calculation of 1:10 and left for a day in the refrigerator (+ 4 ° C). The mixture was then centrifuged with ethyl propyl ether (1 part ether and 3 parts homogenate) for 12 minutes at 5000 rpm in a refrigerator centrifuge twice. The supernatant (445 ml) containing inhibin was placed in a flask in a refrigerator (+ 4 ° C) for a day. Then centrifuged again and determined the total protein in the supernatant (7680 mg), the amount of inhibin-A 95 mg.

Yield 57.9%, degree of purification 2.3.

Ammonium sulfate precipitation

The resulting solution was mixed with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 50 minutes at 10,000 rpm. The resulting precipitate was dissolved in TRIS-HCl buffer with a pH of 7.8. (Total volume 115 ml, total protein 855 mg, amount of inhibin-A 73 mg, yield 44.5%, purification 16.5.)

Affinity chromatography

This stage of purification of inhibin-A is based on the ability of castor bean lectin (ricin) to precipitate inhibin-A that we discovered. Represents inhibin-A affinity chromatography on immobilized castor bean lectin. Through a column (2 × 14 cm) of lectin-sepharose balanced with TRIS-HCl buffer, the entire volume of the dissolved precipitate obtained in the previous step was passed.

Next, the column was washed with a 1 M solution of sodium chloride, buffered with TRIS-HCl buffer, and then the column bound inhibin-A was eluted with a 0.1 M solution of lactose in borate buffer, pH 9.0. The resulting eluate was dialyzed and concentrated to a volume of 13 ml (total protein 32 mg, amount of inhibin-A 27 mg, yield 16.3%, purification 138.8).

Example 3

Primary placenta extract

To prepare the extract, pieces of the placenta were crushed and homogenized by adding saline in a ratio of 1: 2. (Total volume 1200 ml, total protein 31640 mg, inhibin-A 164 mg, 100% yield, purification 1.)

Butanol fraction

To the resulting mass was added butyl alcohol in the calculation of 1:10 and left for a day in the refrigerator (+ 4 ° C). The mixture was then centrifuged with ethyl acetate (1 part ether and 3 parts homogenate) for 11 minutes at 5000 rpm in a refrigerator centrifuge twice. The supernatant (445 ml) containing inhibin was placed in a flask in a refrigerator (+ 4 ° C) for a day. Then centrifuged again and determined the total protein in the supernatant (7680 mg), the amount of inhibin-A 95 mg.

Yield 57.9%, degree of purification 2.3.

Ammonium sulfate precipitation

The resulting solution was mixed with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 50 minutes at 10,000 rpm. The resulting precipitate was dissolved in TRIS-HCl buffer with a pH of 7.8 (total volume 115 ml, total protein 855 mg, amount of inhibin-A 73 mg, yield 44.5%, purification 16.5).

Affinity chromatography

This stage of purification of inhibin-A is based on the ability of castor bean lectin (ricin) to precipitate inhibin-A that we discovered. Represents inhibin-A affinity chromatography on immobilized castor bean lectin. Through a column (2 × 14 cm) of lectin-sepharose balanced with TRIS-HCl buffer, the entire volume of the dissolved precipitate obtained in the previous step was passed. Next, the column was washed with a 1 M solution of sodium chloride, buffered with TRIS-HCl buffer, and then the column bound inhibin-A was eluted with a 0.1 M solution of lactose in borate buffer, pH 9.0. The resulting eluate was dialyzed and concentrated to a volume of 13 ml (total protein 32 mg, amount of inhibin-A 27 mg, yield 16.3%, purification 138.8).

From the above examples it is seen that the yield and degree of purification from the use of different esters do not change. The use of different esters does not affect the final product, since the ester is used to remove lipid fractions and increases the yield of protein components. In our research, we used diethyl ether as the cheapest and most affordable.

A positive effect was achieved: a method was developed that was characterized by a simplification of the technological process, an increase in the yield of the target product, an increase in volumes, a decrease in the number of stages and a decrease in the cost of the target product, and inhibin-A was obtained with a high degree of purification (138.8), increased specific activity and characteristics (see table).

Purification stage The total volume containing inhibin-A Total protein mg The amount of inhibin-A, mg Exit, % Degree of purification Primary placenta extract 1200 ml 31640 164 one hundred one Butanol fraction 445 ml 7680 95 57.9 2,3 Ammonium sulfate precipitation 115 ml 855 73 44.5 16.5 Affinity chromatography 13 ml 32 27 16.3 138.8

Claims (1)

The method of obtaining and purification of inhibin-A, which consists in preparing an extract from biological tissues, sequential precipitation of the protein with organic solvents and ammonium sulfate, by chromatography, characterized in that the pieces of the placenta are crushed and homogenized, physiological saline is added in a ratio of 1: 2 to the resulting mass add butyl alcohol at a rate of 1:10 and leave for a day in the refrigerator at t + 4 ° C, then the mixture is centrifuged with diethyl ether 1: 3 for 10-12 minutes at 5000 rpm at a refrigerated price twice, the inhibin-containing supernatant was placed in a flask in a refrigerator at t + 4 ° C for a day, then centrifuged again and the total protein was determined in the supernatant, the resulting solution was mixed with an equal volume of a saturated solution of ammonium sulfate and centrifuged in a refrigerator centrifuge for 45-50 min at 10,000 rpm, the resulting precipitate was dissolved in TRIS-HCl buffer with a pH of 7.8, applied to a lectin-sepharose column balanced with TRIS-HCl buffer, the entire volume of the dissolved precipitate obtained in previous stage, the column is washed with a 1M solution of sodium chloride, buffered with TRIS-HCl buffer, then the inhibin-A bound with a 0.1M solution of lactose in borate buffer pH 9.0 is eluted, the eluate is dialyzed and concentrated.