Introduction
A failure of homeostasis and consequent formation of blood clots in circulatory system can produce severe outcomes such as stroke and myocardial infarction. Pathological development of blood clots requires clinical intervention with fibrinolytic agent such as urokinase, tissue plasminogen activator and streptokinase (1).
Streptokinase (SK) is a non-protease plasminogen activator, produced by certain streptococci and certain bacteria which contain appropriate genetic material derived from streptococci of Lancefield groups A, C or G. Tillet (2) discovered that this bacterial protein caused the lysis of human blood clots. The fibrinolytic activity of streptokinase originates in its ability to activate blood plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site (3, 4). Due to this property it is used in clinical medicine as a therapeutic agent in the treatment of thromboembolic blockage, including coronary thrombosis.
The mature streptokinase has a molecular weight of about 47 kD and was found to be composed of 415 amino acid residues (5). Secretion of streptokinase into the external medium is directed by a 26 amino acid signal peptide which is cleaved during the secretion process.
Production of natural and recombinant forms of SK and its purification by different chromatography methods, which are based on quantitative differences in solubility, electrical charge, molecular size and shape or non specific physical interactions with surfaces, has been studied by several workers (6, 7). The growth of a β-hemolytic streptococcus was studied in continuous culture with pH as a limiting factor (8, 9).
We have already reported the rate of SK production in a fed-batch culture and purification by affinity chromatography on acylated plasminogen with ρ-nitro phenyl guanidine-benzoate (NPGB) (10). The gene codes for streptokinase, from Streptococcus equisimilis (S.equisimilis) H46A was expressed in several heterologous gram positive and gram negative bacteria (11, 12). A fusion recombinant streptokinase has been produced and purified it in a single step affinity chromatography using glutathione as the ligand (13).
In the present study, the issue of purification of recombinant Streptokinase (rSK) is addressed, for which a new immunoaffinity chromatography approach to produce active rSKC in a single step with high yield and stability was introduced.
Materials and Methods
The materials used in the experiment includes; H46A (ATCC 12449, USA), E.coli DH5α and E.coli BL21 (DE3) pLysS (Invitrogen, USA), Todd Hewitt Broth (THB, HiMEDIA Laboratories), Trypticase Soy Agar (TSA, BBL, USA) Lysine monohydrochloride (Sigma Chemical, USA), pGEX-1.2-4T-2 (Avicenna Research Institute, Iran), Sodium Dodecyl Sulfate (SDS, Sigma Chemical, USA) Hexyl resorcinol (Merck, Germany), NPGB (Sigma Chemical, USA), 3-amino-n-caproic acid (EACA, Sigma Chemical, USA), Chromogenic substrate (S-2251, Chromogenix laboratories, Italy), Isopropyl-beta-D-thiogalactopyranoside (IPTG, Roche, Germany), Cyanogen bromide-activated Sepharose 4B (Sigma Chemical, USA), Sepharose 6MB-Protein A (GE healthcare, USA), Dimethyl pimelimidate-HCL (DMP, sigma-Ald-rich, USA), Phenylmethylsulfonyl fluoride (PMSF, sigma Chemical, USA), serum albumin (sigma-Aldrich, USA), sheep anti-rabbit IgG (Avicenna Research Institute, Iran), o-Phenylenediamine (OPD, sigma Chemical, USA), Polyvinylidine difluride (PVDF, Roche, Germany), Enhanced Chemilumescent substrates (ECL, GE Health care, Biotech Buck-inghamshir, UK), Chemicals for PAGE (Sigma Chemical, USA), Salts for buffers (Merck, Germany).
Preparation of streptokinase-sepharose column
S.equisimilis group C, strain H46A was grown in TSA and THB. The secreted SK purified by protected affinity chromatography (10). The purified streptokinase was then coupled to CNBr-activated Sepharose 4B. Briefly, 5 mg of purified SK was added per ml of the CNBr-activated Sepharose 4B resin in 0.1 M Sodium bicarbonate (NaHCO3, pH = 8.3) and after 2 hr at room temperature, the resin was washed with 0.2 M glycine, (pH = 8) and then alternately for three times with 0.1 M sodium acetate, 0.5 M NaCL, (pH = 4) and 0.1M NaHCO3, (pH = 8.3). The coupled streptokinase-sepharose was transferred to column, neutralized by Phosphate Buffered Saline (PBS) and stored in PBS-azide.
Production and purification of anti-streptokinase
Rabbit was immunized by subcutaneous injection of 1500 µl of an emulsion containing 25 µg of streptokinase and equal volume of complete Freunds adjuvant at 3 weeks intervals. Blood collected and serum was separated. Ten ml of sera was diluted 5 times with PBS, centrifuged at 4000 rpm for 10 min at 4
o
C to remove residual particles, and then was filtered. The supernatant was passed through a SK-Sepharose column at a rate of 12 ml per hour. It was washed by PBS and eluted with 0.2 M Glycine-HCl pH = 2.5. Then it was neutralized by PBS. The purity was confirmed by SDS-PAGE.
Expression tests of fusion GST-SK in E.coli
SDS-PAGE analysis: E.coli BL21 (DE3) pLysS was transformed by pGEX-1.2-4T-2 (pGEX vector with Glutathione S-Transferase (GST) tag and SK gene insert) (13). Bacteria was grown in 5 mL LB broth to an OD of 0.6 (A600 nm) with vigorous agitation at 37°C. Fusion protein expression was induced by 0.1 mM IPTG and incubated for an additional 7 hr. One ml of the culture was collected at two hours intervals (1, 3, 5 and 7 hr), spun down and pellet was resuspended in 100 µl of 1x SDS sample buffer containing (7.5% Tris. base, 2 ml of 10% SDS, 1 ml Glycerol, 2 mg Bromophenol blue, 25 µl 2Mercaptoethanol). The cells were lysed using a strong vortex, heated at 100°C for 5 min, spun down and its supernatant was applied to 10% SDS-poly-acrylamide gel electrophoresis. Proteins were stained with Coomassie blue and the band was visualized by destaining. In order to analyse the fusion GST-SK protein Molecular Weight (MW), the Bio-Rad MW marker, Cat. No. 161-0309 was used.
Western blotting:
Sonicates of pre and post inductions of cloned GST-SK in E.coli and also a purified SK were run on SDS-PAGE. The gel was blotted on PVDF membrane using transfer buffer containing 25 mM Tris (pH = 8.3), 192 mM glycine and 20% methanol at 100 v for 75 min at room temperature. Blotted membrane was blocked with 2.5% skim milk in TBST buffer (0.5 M NaCl, 0.02 M Tris pH = 8.5, 0.05% tween 20) overnight at 4°C. After twice washing with PBS-Tween, membranes were incubated for 1.5 hr at room temperature with 1 µg per ml rabbit antistrep-tokinase in 1% skim milk. After reactions with primery antibody, the membrane was washed three times with PBS-Tween and incubated with peroxidase conjugated sheep anti-rabbit IgG (Avicenna Research Institute) at a 1:2000 dilution in 1% skim milk. The membrane was then washed three times with PBS-Tween. The reaction was developed by ECL for 1 min and scanned.
Preparation of antistreptokinase-sepharose column
Cross-linking of antistreptokinase to protein-A sepharose 6MB:
Twenty mg of purified anti-SK antibody in 10 ml of PBS was mixed with 1 ml of protein A-Sepharose 6MB continuously at 4
o
C and washed twice with excess of PBS. The beads rinsed with 8 ml of 0.2 M borate-NaOH, pH = 8.6 and centrifuged for 1 min at 500×g. The washing was repeated with borate buffer two more times. Anti-SK was cross-linked to protein-A Sepharose by addition of 15.5 mg of DMP in 2 ml of 0.2 M triethanolamin buffer, pH = 8.3. to 1 ml of the coupled bead. After rotation for 30 min at room tempreture, the cross-linking and washing steps were repeated twice to improve cross-linking effeciency. The rest of reactive amino groups were quenched by addition of 50 mmol/l glycine-HCL (pH = 3). The anti-bodies cross-linked to beads were stored at 4
o
C in PBS containig 0.02% sodium-Azide Tween-20 (PBST) until use.
Purification of streptokinase on antistreptoki-nase-sepharose column:
Two ml of 50% slurry of antibody-cross linked to protein A-sepha-rose was equilibrated with 20 ml of binding buffer [10 mM 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid (HEPES)-NaOH, pH = 7, 0.1 M NaCl, 2 mM PMSF]. Ten ml of recombinant streptokinase sample [sonicate of a 6 hours post induction of transformed BL21 (DE3) pLysS] was prepared in binding buffer and added on the bead. The binding occurred for 1 hr to over-night by continuously mixing. The bead was packed in column and sequentially washed with 30 ml of TBS (20 mM Tris-HCl, pH = 7.5. 150 mM NaCl) with a flow rate of 1 ml/min and then with 10 ml pre-elution buffer (0.1 X TBS). The bound proteins were eluted with 5 ml of 50 mM sodium phosphate, pH = 12. Five 1 ml fractions collected into tubes preloaded with 1 ml of cold 1 M Tris-HCl, pH = 6. As soon as each fraction volume reaches 2 ml, contents of each tube were mixed to neutralize the eluate and placed on ice. The column was regenerated immediately after use by washing with 40 ml of 1 M Tris-HCl.
The activity of purified SK was measured by plasmin hydrolysis of chromogenic peptidyl anilide substrate (S-2251), procedure (14) with slight modification by us (13).
Discussion
Purification of SK have already been studied by various methods including; column chromatography on DEAE-cellulose followed by column electrophoresis in sucrose density gradients (15), a combination of ion exchange (DEAE-Sephadex A-50) and gel permeation (Sephadex G-100) chromatography (16). These methods required repeated chromatography to remove the impurities. As a result, the SK recovery yields found to be 40-60%.
In other attempts SK was fractionated by ammonium sulfate and further purified by gradient elution from a DEAE-cellulose chromatography column (17). Several affinity chromatography methods were applied by different workers, including; insolubilized diisopropyl fluorophosphates (DIP) plasmin as the affinity ligand (18). This method caused a 30% decrease in the streptokinase activity.
As mentioned earlier, SK was purified by a protected affinity chromatography method in which the ligand (plasmin) was acylated to protect its activation by SK (10). Even though the yield of purification by this method was found to be 95%, but acylation is not permanent and remains stable just for less than 4 hours. We have produced a recombinant SK tagged by a poly Histidin tail and purified both of the 6xHis-SK and GST-SK by affinity chromatography on NiNTA-agarose and glutathione-sepharose columns respectively, using thrombin for cleavage of the tags (unpublished). Thrombin is a costly enzyme, so this method is not cost effective for large scale purification.
Recently, we have extracted and purified the native SK from H46a fermentation broth by chemical reduction method based on the specific structure of SK, lacking disulfide bridge (19). This method may not be applicable for recombinant SK purification from other bacterial sources, as they may secret proteins lacking disulfide bridges.