Cloning and expression of hybrid streptokinase towards clot-specific activity

Buniya, Harith K.; Murugan, Vadivel; Thangadurai, Chinnathambi

doi:10.1016/j.mimet.2014.01.006

Cited by 6 publications

(6 citation statements)

References 22 publications

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“…The restriction enzymes (NdeI, KpnI, and EcoRI), Taq DNA polymerase, and T4 DNA ligase were obtained from NEB (USA). The positive clones were con rmed by DNA sequencing (Buniya et al 2014).…”

Section: Preparation Of Hybrid Streptokinase Constructsmentioning

confidence: 99%

“…The soluble part in the supernatant was higher, than the insoluble in the pellet, and the KSK protein was expressed as a soluble form better than FSK and FKSK (Figure 2 C). when checked the protein activity, KSK has high activity in colt lysis assay (Buniya et al 2014).…”

Section: Solubility Of Kskmentioning

confidence: 99%

“…Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent in the treatment of acute myocardial infarction (Collen and Lijnen 1991;Lijnen and Collen 2000). Structurally modi ed Streptokinases (non-speci c brin) have been produced in recombinant DNA technology by fused nger and kringle 2 domains from tissue plasminogen activator gene (speci c to brin) with SK gene to produce clot speci c hybrid SK in E.coli as an expression host and T7 expression system (Buniya et al 2014) .…”

Section: Introductionmentioning

confidence: 99%

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Potential E. coli Expression Strategies for Production of Soluble Recombinant Streptokinases

Buniya

2022

Preprint

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The expression of recombinant protein for industrial and therapeutic applications, requires high yielding expression system like E. coli, which is easier to handle and satisfactory for various purposes. On other hand, one of the limitations of this system is Inclusion bodies formation. In this study, we optimized the conditions for the expression of the soluble form for three recombinant streptokinases (FSK, KSK, and FKSK). The change in culture conditions, like optical density, post-induction temperature, inducer concentration, media, and expression host had different effects on the expression levels of hybrid streptokinase proteins. The high amount of soluble fraction for FSK protein was in LB medium at 20°C in BL21 (DE3) and GJ1158. The amount of soluble protein was approximately 50%, but the amount of total protein at 20°C was lower than other induction temperatures. The high amount of soluble protein, expressed hybrid streptokinase (KSK) in LB medium at 30°C in BL21 (DE3) hosts, 0.5 O.D. cell densities were induced in 0.5mM IPTG. Approximately 70% of protein is soluble in these conditions. FKSK was never produced as a soluble form irrespective of any change in condition. All expressed FKSK protein appeared as inclusion bodies.

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Section: Preparation Of Hybrid Streptokinase Constructsmentioning

confidence: 99%

Section: Solubility Of Kskmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Potential E. coli Expression Strategies for Production of Soluble Recombinant Streptokinases

Buniya

2022

Preprint

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“…Using the gene-specific primers SK F1 and SK R1 (Table1) and an annealing temperature of 58 °C, the 1.3-kb SK gene was amplified by PCR (Buniya et al, 2014). The sequence of SK was recorded in the NCBI database.…”

Section: Construction and Molecular Cloning Of The Sk Gene And Vectormentioning

confidence: 99%

“…The wells were filled with 50 microliters of the native SK and left to incubate for 24 hours at 37 °C. It was demonstrated how the wells' clear zone developed (Buniya et al, 2014).…”

Section: Caseinolytic Testmentioning

confidence: 99%

Cloning and expression of native streptokinase in E. coli BL21(DE3)

Hameed

Al-Hayawi

Buniya

2022

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Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria. Streptokinase is one of the most important thrombolytic agents that was frequently employed treatment of myocardial infarction. In this study, we amplified the SK gene isolated from Streptococcus pyogenes. The sequence of SK was recorded in the NCBI database. The Accession number of the SK sequence was LC625519.1. The SK gene was digested by EcoR1 and Hindlll, ligated with the linear pGEM®-3Zf (+) vector, and then introduced into an expression host E. coli BL21(DE3). For the production of the SK protein, we induced by 1mM IPTG. SDS-PAGE was used for the evaluation of the streptokinase synthesis with a molecular weight of around 47 kDa. Using the casein lysis method and the in vitro clot lysis assay, the activity of native SK was assessed. The native SK has clot lysis activity and a clear zone in the casein plate.

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