Novel preparation protocol for the expression and purification of recombinant staphylokinase

Ren, Dongsheng; Li, Dehua; Yang, Weihua; Li, Yun; Gou, Xiaoping; Liang, Bo; Li, Bogang; Wu, Qingping

doi:10.1042/ba20070225

Cited by 8 publications

(3 citation statements)

References 20 publications

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“…Later, it was cloned and expressed to various levels in different expression systems, like Bacillus subtilis, Streptomyces lividans and Pichia pastoris (Ren et al 2008). The aim of our research is to obtain transgenic plants for the production of recombinant staphylokinase (Dobrowolska and Kononowicz 2004).…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…SAK gene has been cloned and expressed to varied levels in different expression systems like Escherichia coli, Bacillus subtilis, Streptomyces lividans, and Pichia pastoris (Nagnath et al 2009;Ren et al 2008;Ye et al 1999;Cheng et al 1998;Miele et al 1999).…”

Section: Introductionmentioning

confidence: 99%

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

et al. 2009

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Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.

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“…Hence, Sak has become a promising drug and stimulated much structural and protein engineering research. To date, sak gene has been cloned and expressed in different expression systems including Escherichia coli (Ren et al, 2008;Mandi et al, 2009), Bacillus subtilis (Ye et al, 1999), Streptomyces lividans (Cheng et al, 1998) and Pichia pastoris (Miele et al, 1999;Apte-Deshpnade et al, 2009), andunder various E. coli promoters viz., T7, lambda PR, tac andptac (Lee et al, 1998), B. subtilis promoters of P43, Pamy and PsacB, and P. pastoris AOX1 promoter.…”

Section: Introductionmentioning

confidence: 99%

Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakC, from Staphylococcus aureus QT08 in Escherichia coli BL21

Nguyễn¹,

Quyen²

2012

Afr. J. Biotechnol.

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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.

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Novel preparation protocol for the expression and purification of recombinant staphylokinase

Cited by 8 publications

References 20 publications

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakC, from Staphylococcus aureus QT08 in Escherichia coli BL21

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