Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakC, from Staphylococcus aureus QT08 in Escherichia coli BL21
Abstract: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.… Show more
The application of recombinant proteins is rare following the high production costs of expressing proteins with expensive inducers, such as isopropyl-β-D-1-thiogalactopyranoside (IPTG). Staphylokinase (SAK), a fibrinolytic enzyme, is a small bacterial thrombolytic agent that specifically clots and converts plasminogens to plasmins and lysis fibrin clots. The primary objective of the present investigation sought to increase the yield and lower the cost of staphylokinase production using Escherichia coli BL21 (DE3). Although the influence of the culture medium, culture density, and IPTG concentration on the production of SAK protein was explored. The results indicated that only culture density and concentration of IPTG were significant. This study achieved cost reduction by decreasing the IPTG inducer concentration (1.0 and 0.5 mM), which acted as the inducer. The production rate was also maintained or increased in low culture density. In conclusion, suitable production conditions, particularly diminished inducer concentration, effectively reduced upstream production costs and yielded high sak gene expression.
The application of recombinant proteins is rare following the high production costs of expressing proteins with expensive inducers, such as isopropyl-β-D-1-thiogalactopyranoside (IPTG). Staphylokinase (SAK), a fibrinolytic enzyme, is a small bacterial thrombolytic agent that specifically clots and converts plasminogens to plasmins and lysis fibrin clots. The primary objective of the present investigation sought to increase the yield and lower the cost of staphylokinase production using Escherichia coli BL21 (DE3). Although the influence of the culture medium, culture density, and IPTG concentration on the production of SAK protein was explored. The results indicated that only culture density and concentration of IPTG were significant. This study achieved cost reduction by decreasing the IPTG inducer concentration (1.0 and 0.5 mM), which acted as the inducer. The production rate was also maintained or increased in low culture density. In conclusion, suitable production conditions, particularly diminished inducer concentration, effectively reduced upstream production costs and yielded high sak gene expression.
This study was aimed to purify, characteristic, and fibrinolytic activity of staphylokinase (SAK), is an enzyme activates plasminogen to form plasmin, which digest fibrin clots that cause thrombosis clot. Staphylokinase was purified from local isolate Staphylococcus aureus GH38 by ammonium sulfate precipitation at 70% saturation followed by ion exchange chromatography (CM-Cellulose) with a purification fold 2.73, and 1.53, and recovery 72.1, and 33.11% in wash and elution steps respectively. The partially purified enzyme was high activity at 40°C with pH 7 and the enzyme retained 100% of its activity at 35°C with pH 7. The activity of an enzyme increased by its treatment with calcium and sodium chloride, while the activity affected when incubated with mercury, silver, and iron chloride. The enzyme have high effective against thrombus (blood clot), which encourage the use of enzyme in the treatment as therapeutic agent to remove clots formed in the human body.
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