Abstract:The effect of several ions (Cl -, Na + , K + , Ca 2+ ) on the rate of plasminogen (Pg) activation by recombinant staphylokinase (rSTA) is reported. Both monovalent and divalent ions affect the rate at which Pg is activated by rSTA, in a concentration-dependent manner (range 0-100 mM). In almost all cases, a decrease of the initial velocity of activation was observed. Cl -showed the most striking inhibitory effect at low concentrations (64% at 10 mM). However, in the presence of a fibrin surface, this inhibitio… Show more
“…In contrast, Ca 2+ and Mg 2+ showed an opposite effect on the rSak activity in this study and the native Sak activity studied by Vesterberg and Vesterberg (1972). The same results is also found in the report that Cl -, Na + , K + and Ca 2+ affected the rate at which plasminogen was activated by rSak (Yarzábal et al, 1999).…”
The staphylokinase (Sak) is emerging as an important thrombolytic agent for the treatment of patients suffering from cardiovascular disease. Hence in this study, we reported the cloning, high-level expression, purification and characterization of the Sak variant Sakφ φ φ φC from Staphylococcus aureus QT08 in Escherichia coli Bl21. The sak gene of 489 bp encoding a protein (163 amino acids) with a predicted molecular mass of 18.5 kDa and pI 7.28 showed 99.8 to 99.6% identity with corresponding sequences from S. aureus strains deposited in GenBank (AF332619, X00127, EF122253 and M57455). The DNA sequence (411 bp) encoding the mature Sak (15.5 kDa) truncated 27 N-terminal amino acids was expressed in E. coli BL21/pESak under the control of the strong promoter tac in the presence of isopropyl-β-D-1-thiogalactopynoside (IPTG) as inducer. The expression level of rSak was estimated at about 42% of the total cellular proteins by densitometry scanning, which is the highest expression level of rSak expressed in any E. coli system. The recombinant staphylokinase was purified by Ni 2+ -ProBond™ ™ ™ ™ column to a single homogeneous 16-kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a specific activity of 15175 U/mg protein, a recovery yield of 58% and a purification factor of 2.56. The optimal pH and temperature for the rSak activity was 9 and 37°C, respectively. rSak was stable over a temperature range of 25 to 50°C and at pH range of 7 to 9. Metal ions and detergents also showed an inhibitory effect on rSak, especially Zn 2+ and Cu 2+ which completely inhibited the enzymatic activity.
“…In contrast, Ca 2+ and Mg 2+ showed an opposite effect on the rSak activity in this study and the native Sak activity studied by Vesterberg and Vesterberg (1972). The same results is also found in the report that Cl -, Na + , K + and Ca 2+ affected the rate at which plasminogen was activated by rSak (Yarzábal et al, 1999).…”
The staphylokinase (Sak) is emerging as an important thrombolytic agent for the treatment of patients suffering from cardiovascular disease. Hence in this study, we reported the cloning, high-level expression, purification and characterization of the Sak variant Sakφ φ φ φC from Staphylococcus aureus QT08 in Escherichia coli Bl21. The sak gene of 489 bp encoding a protein (163 amino acids) with a predicted molecular mass of 18.5 kDa and pI 7.28 showed 99.8 to 99.6% identity with corresponding sequences from S. aureus strains deposited in GenBank (AF332619, X00127, EF122253 and M57455). The DNA sequence (411 bp) encoding the mature Sak (15.5 kDa) truncated 27 N-terminal amino acids was expressed in E. coli BL21/pESak under the control of the strong promoter tac in the presence of isopropyl-β-D-1-thiogalactopynoside (IPTG) as inducer. The expression level of rSak was estimated at about 42% of the total cellular proteins by densitometry scanning, which is the highest expression level of rSak expressed in any E. coli system. The recombinant staphylokinase was purified by Ni 2+ -ProBond™ ™ ™ ™ column to a single homogeneous 16-kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a specific activity of 15175 U/mg protein, a recovery yield of 58% and a purification factor of 2.56. The optimal pH and temperature for the rSak activity was 9 and 37°C, respectively. rSak was stable over a temperature range of 25 to 50°C and at pH range of 7 to 9. Metal ions and detergents also showed an inhibitory effect on rSak, especially Zn 2+ and Cu 2+ which completely inhibited the enzymatic activity.
Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth, respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35 °C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40 °C. In addition it retained 100% of its activity at temperature ranged between 15 and 45 °C and pH ranged from pH 6.0 to 9.0. EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.
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