High yielding recombinant Staphylokinase in bacterial expression system—cloning, expression, purification and activity studies

Mandi, Nagnath; Soorapaneni, Sudheerbabu; Rewanwar, Sachin; Kotwal, Prakash; Prasad, Bhaskarjyoti; Mandal, Gautam; Padmanabhan, Sriram

doi:10.1016/j.pep.2008.10.010

Cited by 23 publications

(18 citation statements)

References 20 publications

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“…There are several reports indicating that the estimated cost of producing recombinant proteins in plants may be 10-15 times lower compared to the synthesis of the same product using the E. coli bacterial system (Giddings 2001). Staphylokinase, a protein of bacterial origin, belongs to plasminogen activators that are considered as potential therapeutics for thrombolytic therapy (Mandi et al 2009;Moreadith and Collen 2003;Bokareva et al 2006). In this paper, we report for the first time, transgenic Arabidopsis thaliana plants that express a staphylokinase coding sequence fused to gusA and mgfp reporter genes.…”

Section: Western Blot Analysis In T1-generation Of a Thaliana Plantsmentioning

confidence: 99%

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Wiktorek-Smagur

Hnatuszko-Konka

Geszberg

et al. 2010

World J Microbiol Biotechnol

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A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta Agrobacterium tumefaciens-mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated plants. The presence of the fusion protein (119 kDa) was confirmed by SDS-PAGE and Western blot analysis in protein extracts from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of 45 transgenic lines of A. thaliana plants.

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Section: Western Blot Analysis In T1-generation Of a Thaliana Plantsmentioning

confidence: 99%

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Wiktorek-Smagur

Hnatuszko-Konka

Geszberg

et al. 2010

World J Microbiol Biotechnol

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“…One notable exception is when transforming with plasmid constructs containing recombinant genes under control of the T7 polymerase, these constructs are typically transformed into DH5 cells during the cloning stage and later introduced into a bacterial strain expressing T7 RNA polymerase for expression of the recombinant protein. The derivatives available for this purpose include BL21(DE3), BL21A1 which are all lon and OmpT protease negative strains (Banerjee et al, 2009;Mandi et al, 2008) and ER2566 (Yu et al, 2004) strains.…”

Section: Types Of E Coli Host Cells Used For Transformationmentioning

confidence: 99%

Screening of Bacterial Recombinants: Strategies and Preventing False Positives

Padmanabhan¹,

Banerjee²,

Mandi³

2011

Molecular Cloning - Selected Applications in Medicine and Biology

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“…The SAK activity was performed using a chromogenic substrate assay as reported by Mandi et al, (2009) while the antiviral activity was done using WISH cells and Encephalomyocarditis virus challenge method by cytopathic effect method and using standard NIBSC interferon preparation at National Institute of Virology, Pune, India as per the published protocol (Yousefi et al, 1985 Figure 1 shows the PCR product of IFNα2b from the synthetic DNA. While the results shown in Figure 2a and 2b show the plasmid map of pET21a-IFN clone and small scale expression of the IFN from pET21a-IFN in BL21A1 (without rare codon supplementation) and in BL21(DE3) codon plus cells (with rare codon supplementation) respectively.…”

Section: Sak Activity and Ifnα2b Anti-viral Activitymentioning

confidence: 99%

Expression and Purification of SAK-fused Human Interferon Alpha in Escherichia coli

Salunkhe¹,

Prasad²,

Sabnis-Prasad³

et al. 2009

J Microbial Biochem Technol

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A method for improved refolding and purification of E. coli derived human Interferon -α (rhIFN α2b) from inclusion bodies as a Staphylokinase (SAK) fusion protein is described. Such a fusion protein did not require the supplementation of rare codons for expression and was found to be stable at 37 o C. The optimal conditions of refolding involved the use of a mild denaturating agent without the need for any other agents to prevent aggregation. The SAKrhIFN α2b fusion protein was successfully purified using two steps of purification and was cleaved using enterokinase into two fragments namely SAK and IFN. Both the proteins were found to be biologically active showing proper folding of both the fusion partners. The cleaved IFN showed similar retention time on RP-HPLC as the bacterial derived untagged purified IFN as well as similar molecular weight on Agilent 2100 Bioanalyzer indicating the right processing of the IFN after enterokinase cleavage. The expression levels of SAK-IFN were found to be two folds higher than that observed with untagged IFN under similar experimental conditions.

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High yielding recombinant Staphylokinase in bacterial expression system—cloning, expression, purification and activity studies

Cited by 23 publications

References 20 publications

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Screening of Bacterial Recombinants: Strategies and Preventing False Positives

Expression and Purification of SAK-fused Human Interferon Alpha in Escherichia coli

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