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Production and purification of streptokinase by protected affinity chromatography.

Introduction

The clinical importance of streptokinase was first noted by Tillet and Garner (1), who discovered that this bacterial protein caused the lysis of human blood clots. It was later found that streptokinase is not an enzyme but rather a potent activator of plasminogen, the inactive precursor of plasmin (2,3). Plasmin is the active fibrinolytic component of the circulatory system, solubilizing the fibrin network in blood clots through limited proteolysis (4,5).

Streptokinase is currently used in clinical medicine as a therapeutic agent in the treatment of thromboembolic blockages, including coronary thrombosis (6,7). Streptokinase is naturally produced and secreted by various strains of hemolytic streptococci. The best studied of these is the streptokinase from Streptococcus equisimilis, from which the secretion of streptokinase into the external medium is directed by a 26 amino acid signal peptide which is cleaved during the secretion process. The mature protein has a molecular weight of about 47 kilo Dalton (kD) and was found to be composed of 415 amino acid residues (8). Karush, Iacocca, and Harris (9) and Ogburn, Harris, and Harris (10) studied the growth of a [beta]-hemolytic streptococcus in continuous culture with pH as a limiting factor. In these experiments, pH was controlled only by addition of buffer to the medium. The yield of cells and of some extra cellular antigens was investigated. Rosenberger and Elsden (11) studied the effect of both glucose and tryptophan limitation on growth in continuous cultures of a Streptococcus faecalis strain. Their findings indicate that, to obtain maximal cell yield per unit energy source, the energy source should be the limiting factor.

Several methods have been reported for the purification of streptokinase obtained from the culture media of various strains of streptococci. In some cases DEAE-cellulose has been used in combination with other purification procedures (12,13,14) and a highly purified product has been obtained (13).

Other chromatographic procedures have also been used for the purification of streptokinase by combining more than one purification step (15,16). Castellino et al (15) reported the use of affinity chromatography on immobilized Di-Isopropyl phosphate (DIP)-plasmin for single step purification of streptokinase. This method involved the conversion of plasminogen to plasmin by urokinase and the inhibition of plasmin protease activity by diisopropyl fluorophosphates.

Jeong et al (17) reported an affinity chromatography using plasminogen as a ligand. Recently we have produced a fusion recombinant streptokinase and purified it in a single step affinity chromatography using glutathione as the ligand (18).

In this paper, we report the results obtained from the cell growth in Todd Hewitt Broth (THB) culture media supplied with excess glucose at optimum pH and temperature; included in this report is also the rate of streptokinase production and purification by affinity chromatography on acylated plasminogen with [rho]-nitro phenyl guanidinobenzoate (NPGB).

Materials and Methods

The materials used in the experiment include; Streptococcus equisimilis group C, strain H46A (ATCC 12449, USA), Todd Hewitt Broth media (THB, HiMEDIA Laboratories), Trypticase Soy Agar (TSA, BBL, USA), Lysine monohydrochloride (Sigma Chemical, USA), Hexyl resorcinol (Merck, Germany), [rho]-nitro phenyl guanidinobenzoate (NPGB, Sigma Chemical, USA), 3-amino-n-caproic acid (EACA, Sigma Chemical, USA), Cyanogen bromide activated Sepharose 4B (Sigma Chemical, USA), Chromogenic substrate S-2251 (Chromogenix laboratories, Italy), Buffer salts, acids and bases (Merck, Germany).

Extraction of streptokinase from H46A culture

The bacteria were cultured in TSA at 37[degrees]C. One of the colonies was grown in 25 ml of THB at 37[degrees]C. By increasing the turbidity to the level of OD=0.6 at 600 nm, it was sub-cultured in 250 ml of broth; the activity of secreted streptokinase was determined by solid and liquid colorimetric methods (19,20). It was observed that the optimum PH for cell growth and streptokinase activity was at the neutral condition (pH=7). To improve the growth condition, the pH of the culture was maintained at 7 during incubation at 37[degrees]C for 8 hours by adding sterile 4% (w/v) glucose and 5.0 N NaOH. The culture was centrifuged for 25 minutes at 10,000 g. Prior to addition of solid ammonium sulfate to a final concentration of 65% (w/v), the supernatant was filtered through a 0.45 [micro]m cellulose acetate filter. After standing at 4[degrees]C overnight, the precipitate was harvested by centrifugation at 4[degrees]C for 20 minutes at 12,000 g and dissolved in 1 ml of 10 mM Tris buffer, pH=8.0, and dialyzed against repeated changes of the same buffer.

Preparation of plasminogen affinity column

Purified plasminogen was prepared from human plasma by lysine Sepharose affinity chromatography (21). 500 ml of human plasma was centrifuged at 3000 rpm for 1 hour at 4[degrees]C to remove residual particles present in the plasma.

The supernatant then was diluted to one liter in 0.003 M EDTA and passed through a 50 ml lysine-Sepharose column at a rate of 70 ml per hour and then washed with 0.3 M sodium phosphate at pH=7.4. Upon elution with 0.3 M sodium phosphate, 0.2 M EACA, pH= 7.4, fractions of 4 ml were collected.

The material was dialyzed against 0.1 M NaHCO3, pH=8.3 and lyophilized. The purity of plasminogan was confirmed by SDS- PAGE (Figure 1). The final yield of the process was approximately 67 mg plasminogen per 500 ml of plasma.

[FIGURE 1 OMITTED]

Approximately 6 mg plasminogen was coupled per ml of the cyanongen bromide-activated Sepharose 4B gel. Plasminogen was then added to the resin in 0.1 M NaHCO3 (pH=8.3), and after 2 hours at room temperature, the resin was washed with 0.2 M glycine, (pH=8.0), and then alternately for three times with 0.1 M sodium acetate, 0.5 M NaCL, (pH=4.0), and 0.1 M NaHCO3, (pH=8.3).

Purification of streptokinase

To purify the streptokinase, a 12 ml bed column of plasminogen coupled to cyanogen bromide- activated sepharose 4B was equilibrated with o.ol M Tris- HCl (pH=8.0). The immobilized plasminogen was treated with 6 ml of 0.5 mM NPGB in 0.01 M Tris-HCI, (pH= 8.0). The NPGB was initially dissolved in dimethylformamide at a concentration of 250 mM.

The dialyzed extract of streptokinase passed through column and the column was then washed with 32 ml of buffer containing 0.01 M Tris-HCI, I M NaCL, (pH= 8.0). Elution was done with 4 ml of 8 M urea in 0.01M Tris-HCL, (pH=8.0).

Results

The rate of streptokinase secretion increased significantly in condition of excess glucose addition to culture media as a result of glucose metabolism and acid production.

Later by neutralizing the acidity with NaOH, the activity of streptokinase increased (Figure 2). The results of the plasminogen (plg) purification are shown in Table 1.

[FIGURE 2 OMITTED]

The streptokinase was purified with and without plasminogen acylation. In the first experiment, we treated the immobilized plasminogen with the specific inhibitor NPGB (Table 2). Two milligram (mg) pure streptokinase was produced, the final product had specific activity of 249850 IU/mg. In an SDS-PAGE analysis, the eluted product showed two bands of approximately 47 and 45 kD (Figure 3, lane 2).

[FIGURE 3 OMITTED]

Discussion

Results from batch cultures indicated that strain H46A produced relatively high yields of streptokinase when the pH in the culture was controlled within the range of 7.0 to 7.1. At pH levels lower than 6.5 or higher than 7.8, streptokinase production decreased to less than 25% of that obtained at neutral pH. The comparison of this study with other studies shows that the optimum pH for cell growth and streptokinase production is 7. By adjustment of culture period, glucose feeding and pH maintances with concentrated NaOH the rate of product increases up to three times.

Moreover, by using suitable amount of hexyl resorcinol, the probable infection with the pathogenic streptococcus during the process was prevented.

In NPGB acylation of the immobilized plasminogen, the NPGB binds covalently to the potential substrate binding site of plasminogen, thus the active site is incapable of hydrolyzing the substrate. As a result, only 5% of the salt extracted streptokinase activity was lost during the process, thus recovering 95% of the total activity (Table 2). As shown in Figure 3, the elution from the column with immobilized acylated plasminogen (lane 2) shows two bands of 47 and 45 kD that corresponds to the streptokinase molecule isotypes secreted by the microorganism. These kinds of streptokinases have affinity with the Glue plasminogen, which is used as a ligand in affinity chromatography.

In the experiment where the immobilized plasminogen was not acylated with NPGB (Table 2), 10% of the streptokinase activity was lost during the process, and the final recovery was of 130%. This increase in streptokinase activity is probably due to the elution of partially degraded plasminogen that forms a stable complex with streptokinase with higher specific activity. Jeong et al reported that recovery of streptokinase in their affinity chromatography on immobilized plasminogen to be about 64%. The results of this study show that the demonstrated purification procedure for streptokinase on NPGB acylated immobilized plasminogen, allows the obtainment of non-degraded products with high specific activity in a single purification step. This way, the method employing acylated plasminogen permits one to obtain a highly purified streptokinase with high yield. The affinity gels may be reused many times without any apparent loss of binding capacity, although NPGB reaction is repeated before each use as a precaution against additional active plasminogen and plasmin being regenerated. This acylation remains stable for less than 4 hours under the conditions described above. We suggest a further study to get an acylation with longer stability or a complete blockage of the immobilized plasminogen binding site.

Acknowledgment

We would like to thank the Iranian Management and Programming Organization for financial support (Grant No: 31309332).

Received: 14 Dec 2008

Accepted: 11 April 2009

References

(1.) Tillet WS, Garner RL. The fibrinolytic activity of hemolytic streptococci's. J Exp Med 1933;58(4): 485-502.

(2.) Schick LA, Castellino F. Direct evidence for the generation of an active site in the plasminogen moiety of the streptokinase-human plasminogen activator complex. J Biochem Biophys Res Commun 1974;57(1):47-54.

(3.) Hoffman R, Benz EJ Jr, Shattil SJ. Hematology: Basic principles and practice (Eds). New York: Churchill Livingstone;1991.

(4.) Rodriguez P, Fuentes P, Barro M, Alvarez JG, Munoz E, Collen D, et al. Structural domains of streptokinase involved in the interaction with plasminogen. Eur J Biochem 1995;229(1):83-90.

(5.) Hermentin P, Cuesta-Linker T, Weisse J, Schmidt KH, Scheld M, Thimme. M. Comparative analysis of the activity and content of different streptokinase preparations. Eur H J 2005;26(9):933-940.

(6.) Banerjee A, Chistic Y, Banerjee UC. Streptokinase, a clinically useful thrombolytic agent. Biotechnol Adv 2004;22(4):287-307.

(7.) Erdogan S, Ozer AY, Volkan B, Caner B, Bilgili H. Thrombus localization by using streptokinase containing vesicular systems. Drug Deliv 2006;13 (4):303-309.

(8.) Jackson KW, Tang J. Complete amino acid sequence of streptokinase and its homology with serine proteases. Biochemistry 1982;21(26):6620-6625.

(9.) Karush F, Lacocca VF, Harris TN. Growth of group a hemolytic streptococcus in the steady state. J Bacteriol 1956;72(3):283-294.

(10.) Ogburn CA, Harris TN, Harris S. Extra cellular antigens in steady-state cultures of the hemolytic streptococcus: Production of proteinase at low pH. J Bacteriol 1958;76(2):142-151.

(11.) Rosenberger RF, Elsden SR. The yields of Streptococcus faecalis grown in continuous culture. J Gen Microbiol 1960;22:726-739.

(12.) Blatt WF, Segal H, Gray JL. Purification of streptokinase and human plasmin and their interaction. Thromb Diath Haemorrh 1964;11:393-403.

(13.) De Renzo EC, Siiteri PK, Hutchings BL, Bell PH. Preparation and certain properties of highly purified streptokinase. J Biol Chem 1967;242(3): 533-542.

(14.) Dillon HC Jr, Wannamaker LW. Physical and immunological differences among streptokinases. J Exp Med 1965;121;351-371.

(15.) Castellino FJ, Sodetz JM, Brockay WJ, Siefring GE. Streptokinase. Methods Enzymol 1976;45:244-257.

(16.) Taylor FB, Botts J. Purification and characterization of streptokinase with studies of streptokinase activation of plasminogen. Biochemistry 1968;7(1):232-242.

(17.) Jeong IK, Young EL, Jeung SJ, Myung B. Purification of streptokinase by affinity chromatography using human plasminogen. Korean Biochem J 1993;26(2):172-175.

(18.) Nejadmoghaddam MR, Modarresi MH, Baba shamsi M, Chamankhah M. Cloning and over expression of active recombinant fusion streptokinase: a new approach to facilitate purification. Pak J Biol Sci 2007;10(13):2146-2151.

(19.) Babashamsi M. Immobilization of plasminogen in agarose for rapid detection of streptokinase. Iranian Biomed J 2000;4:170.

(20.) Jackson KW, Esmon N, Tang J. Streptokinase and staphylokinase. Methods Enzmol 1981;80:387-394.

(21.) Deutsch DG, Mertz ET. Plasminogen purification from human plasma by affinity chromatography. Science 1970;170(962):1095-1096.

Mohammad Babashamsi [1] *, Mohammad Hossein Razavian [2], and Mohammad Reza Nejadmoghaddam [3]

[1.] Department of Medical Diagnostics & Biological Products, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

[2.] Islamic Azad University, Qom branch, Qom, Iran

[3.] Department of Recombinant Technology, Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

* Corresponding author:

Mohammad Babashamsi, Ph.D., Department of Medical diagnostics & Biological products, Monoclonal antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, P.O. Box: 19615-1177

Tel: +98 21 22432020

Fax: +98 21 22432021

E-mail: babashams@avicenna.ac.ir
Table 1. Plasminogen purification results

                             Fraction
Parameters
                      Plasma (PPP)   Result
Total volume (ml)         500          10
Protein (mg)             39500         68
         mg               100          68
Plg      %               0.25          100
         % Recovery      100           67

Table 2. Purification of streptokinase.

     Steps              Streptokinase(IU)

Culture (250ml)            789000
Salt extraction            526000
Purification      On-acylated   Non-acylated
                  Plasminogen   Plasminogen

Washing             26300         52600
Elution            499700         661000
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Title Annotation:Original Article
Author:Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza
Publication:Avicenna Journal of Medical Biotechnology (AJMB)
Article Type:Report
Geographic Code:7IRAN
Date:Apr 1, 2009
Words:2287
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