RU1809387C - Method of purification of @@@-proteinase inhibitor - Google Patents

Abstract

Field of use: medicine, biologists and can be used in the industrial production of protein preparations and medical diagnostics. The essence of the method consists in sequentially carrying out affinity chromatography on blue agarose, covalent chromatography on organo-mercury agarose and metal-chelate chromatography on iminodiacetic acid agarose, which is in copper form. The method can significantly increase the yield of this protein during its purification from the blood plasma of donors, as well as reduce the overall duration of the biotechnological cycle.

Description

. The invention relates to medicine, biology, namely biochemistry, and can be used in the industrial production of protein preparations and medical diagnostics. The aim of the invention was to develop a highly effective method for purifying PI from human blood plasma.

The goal is achieved in that, at the first stage of purification, human blood plasma is passed through a blue agarose gel; fractions containing PI are collected and di-. are assayed against the application buffer, then affinity chromatography on organo-mercury agarose is carried out, with which the PI covalently binds and is subsequently separated by dithiothreitol. The final purification of IP occurs on the copper form of iminodiacetic acid agarose.

The principal differences of the proposed method and its novelty are that in stage I, PI is separated from most of serum albumin, its preparation, as a result, does not contain other proteinase inhibitors (antithrombin III, alpha2-macroglobulin, antichymotrypsin), as well as a number of other plasma proteins, while the loss of PI itself does not exceed 5% (3). At the second stage of purification, PI molecules containing free SH-rpynny cysteine bind highly selectively to mercury, which is part of the organo-mercury sorbent. Most other whey proteins have no free SH groups in the structure of the molecule. Dithirtreitol then, under sufficiently mild conditions, breaks the covalent bond which has arisen. Therefore, on this. at the stage of loss of PIs, they do not exceed 25% (about 5% PIs do not interact at all with organorthutic agarose; therefore, only 20% of protein is lost upon contact with the matrix). At the last stage of purification, PI is more strongly bound to the copper form of iminodiacetic acid agarose than mercaptoalbumin and other mercaptoproteins; therefore, we have an immunochemically pure protein at the output, and its loss here is only 12%. Thus, the total loss of PI during the entire purification does not exceed 37.5%, and the protein yield is 62.5%, which is almost 2 times greater than in the prototype method. In addition, the proposed biotechnological cycle of purification of IP does not exceed 3 days, which is more than 2 times shorter; than in the prototype method.

The zonal electrophoresis in the gradient of pores of the polyacrylamide gel and the electrophoresis according to the Laemmli method gave a molecular mass of 53 KDa, and there were no extraneous proteins in its preparation.

During cross-immuno-electrophoresis with polyserum against all human plasma proteins, the only immunoprecipitate was obtained, which possesses the mobility of alpha-globulins. The PI isolated by us under immuno-diffusion conditions reacted with the monospecific antiserum of the anti-alpha1 proteinase inhibitor from Sigma (USA). The IP purified by us in equimolar pHM with trypsin (Sigma, United States) reduced its proteolytic activity (azocasein substrate) by 95.%, and the amidase activity (Y-benzoyl substrate 01-arginyl-paranit 15 roanilide) by more than 85 % These data indicate a significant nativeness of the protein obtained by us.

. The absence of such methods for purification of PIs, which would ensure a significant yield of native protein of high purity along with a reduction in the duration of the entire biotechnological cycle, indicates the correspondence. technical, solving criteria

25 novelty and significant differences.

Example 1. 150 ml of human blood plasma is dialyzed over 4 ° C against 4 liters of 0.03 M phosphate buffer, pH 7.0. The dialysate is then passed at a rate of 20 ml / h through blue agarose gel. (Estar Small Enterprise, ESSR) having a volume of 400 ml and is in the column. 2.5 cm x LLP cm. Pigeon agarose is pre-balanced with the above

35 buffer. The collection of fractions is carried out

. collector DJAFRACD-002 (NGO Diagnosticum. Lviv), the volume of each fraction

. 10 ml Since PI with blue agarose

practically does not interact, fractions,

40 containing it belong to the earliest and are revealed by the Veremeenko method (7), based on the ability of PIs to inhibit trypsin hydrolysis of 1 1-benzoyl-O1: arginyl-paranitroanilide (BAPNA). These fractions coalesce and dialyze over

 overnight against 4 liters of 0.01 M Tris-hydrochloric acid buffer, pH 7.0, containing 0.01 M

Trilon B. 0.1 M sodium chloride, 0.1%

Sodium dodecyl sulfate. The indicated buffer 50 is pre-equilibrated with a 2 cm x 20 cm chromatography column filled with .50 ml organometrically agarose (Estar Small Enterprise, ESSR). Diali: then passes through this column with

55 at a rate of 2 ml / h. The washing of the organo-mercury agarose is carried out with the initial equilibration buffer to zero optical density (E 280 nm). Elution is accomplished with a well-defined buffer supplemented with 0.01 M dithiothreitol (Reanal,

Hungary). The collection of protein fractions is carried out under the control of a spectrophotometer (E 280 nm). These fractions are collected and dialyzed overnight against four liters of 0.02 M phosphate buffer, pH 8.0, containing 1 M sodium chloride, which serves as a starting buffer for subsequent metal chelate chromatography. 10 ml of iminodiacetic acid agarose (Estar Small Enterprise, ESSR) was placed in a 2. cm x 5 cm column and the gel was transformed into a copper-chelate form (copper is bivalent) as follows. Initially, it is washed with 50 ml of a 0.05 M solution of Trilon B prepared on the basis of a 0.5 M solution of sodium chloride, then it is washed with 50 ml of distilled water, then with 50 ml of a 0.05 M solution of copper acetate. This is followed by washing with 50 ml of distilled water. And equilibrating the gel with the above starting phosphate buffer. The dialysate is passed through the activated gel at a rate of 10. ml / hour. Further; the column is washed with start buffer to zero optical density, Mercaptoalbumin and other impurity proteins are removed with 0.02 M sodium phosphate buffer, pH 6.0, containing 1 M sodium chloride. After completion

 IP gel washes elute with 0.02; M buffer N32HP04 - citric acid. pH 5.0 containing 1 M sodium chloride. It should be noted that the Estar small business matrices used in the invention. (Tartu, ESSR) manufactured according to the temporary technical specifications of the enterprise / which is a trade secret.

.. In order to check the purity of IP, pore gradient polyacrylamide gel electrophoresis, zonal electrophoresis using the Laemmli method, immunoelectrophoresis with antiserum against all serum proteins were performed.

human blood flow (NIIEM, Nizhny Novgorod). In all these tests, the only single chain protein with a molecular weight of 53 K Yes was detected, which possesses the alpha electrophoretic mobility of globulins. The PI isolated by us reacted with a monospecific anti-serum against alpha proteinase inhibitors (Sfgma, USA). In functional tests, PI almost completely suppressed amidase and proteolytic. trypsin activity (more than 85% and 95%, respectively). Thus, the naivety and high degree of purification of the preparation. The IP is proved. .. . Example 2. We isolated PI used for immunization of rabbits. Immunization was carried out according to standard schemes for T years. Even after 10 immunizations, rabbits produced monospecific antibodies against the protein in question. This is another proof of the high degree of purity and nativeness of the IP. . .d. .

FORMULA AND SECTION

 4 .

The method of purification of an en-inhibitor of proteases from human blood plasma using metal chelate chromatography, and so on, and so that, in order to increase the purification efficiency, the blood plasma is subjected to the first step affinity chromatography on blue agarose at pH 7.0; in the second step, covalent chromatography on organo-mercury agarose is carried out in the presence of 0.1% sodium dodecyl sulfate, 0.01 M trilon B at pH 7.0, and then copper (II) - iminodiacetic agarose chelate chromatography in the presence of 1 M sodium chloride at pH 8.0.