Limits...
Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities.

Chen H, Mo W, Su H, Zhang Y, Song H - BMC Mol. Biol. (2007)

Bottom Line: Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK.Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Dong' an Road 130#, Fudan university, Shanghai, 200032, PR China. chenhongshan999@sohu.com

ABSTRACT

Background: Although staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK).

Results: By using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.

Conclusion: RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.

Show MeSH

Related in: MedlinePlus

Production of an engineered staphylokinase-based RGD-SAK. (A) Amino acid residues and nucleotide sequences of SAK and RGD-SAK are shown. The lys35 has been substituted with Arg to constitute a RGD motif and sequence analysis confirmed the successful mutagenesis. (B) Coomassie blue-stained 15% SDS-PAGE showing expression of RGD-SAK. Lane M, protein marker; lanes 1–6, induced for 0, 0.5, 1, 1.5, 2, 2.5 h. (C) Purification of recombinant protein RGD-SAK. Lane M, protein marker; lane 1, cytosol fractions; lane 2, purification after SP-Sepharose; lane 3, purification after Sephadex G-50; lane 4, purification after Q-Sepharose. (D) Western blot analysis with polyclonal anti-SAK antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2194782&req=5

Figure 1: Production of an engineered staphylokinase-based RGD-SAK. (A) Amino acid residues and nucleotide sequences of SAK and RGD-SAK are shown. The lys35 has been substituted with Arg to constitute a RGD motif and sequence analysis confirmed the successful mutagenesis. (B) Coomassie blue-stained 15% SDS-PAGE showing expression of RGD-SAK. Lane M, protein marker; lanes 1–6, induced for 0, 0.5, 1, 1.5, 2, 2.5 h. (C) Purification of recombinant protein RGD-SAK. Lane M, protein marker; lane 1, cytosol fractions; lane 2, purification after SP-Sepharose; lane 3, purification after Sephadex G-50; lane 4, purification after Q-Sepharose. (D) Western blot analysis with polyclonal anti-SAK antibodies.

Mentions: Using PCR-mediated site-directed mutagenesis, the SAK variant RGD-SAK was constructed with Arg in place of K35, and the desired mutant was identified by DNA sequencing (Fig. 1A). After temperature induction, RGD-SAK was over-expressed due to the temperature inducible pL and pR promoter in the expression vector pLY-4. SDS-PAGE showed that the target protein was expressed after 0.5 h of induction and reached peak after 2.5 h (Fig. 1B). The protein was isolated by homogenization and purified by sequential chromatography through SP-Sepharose, Sephadex G-50, and Q-Sepharose (Fig. 1C). High purity protein was obtained (over 98% by densitometric scanning). Western blot (Fig. 1D) revealed that the RGD-SAK protein had a similar antigen binding capacity to SAK. Using known concentrations of pure SAK as the standard, the yield per liter culture of purified RGD-SAK was estimated to be 0.3 g and more than 60% was recovered in purification.


Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities.

Chen H, Mo W, Su H, Zhang Y, Song H - BMC Mol. Biol. (2007)

Production of an engineered staphylokinase-based RGD-SAK. (A) Amino acid residues and nucleotide sequences of SAK and RGD-SAK are shown. The lys35 has been substituted with Arg to constitute a RGD motif and sequence analysis confirmed the successful mutagenesis. (B) Coomassie blue-stained 15% SDS-PAGE showing expression of RGD-SAK. Lane M, protein marker; lanes 1–6, induced for 0, 0.5, 1, 1.5, 2, 2.5 h. (C) Purification of recombinant protein RGD-SAK. Lane M, protein marker; lane 1, cytosol fractions; lane 2, purification after SP-Sepharose; lane 3, purification after Sephadex G-50; lane 4, purification after Q-Sepharose. (D) Western blot analysis with polyclonal anti-SAK antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194782&req=5

Figure 1: Production of an engineered staphylokinase-based RGD-SAK. (A) Amino acid residues and nucleotide sequences of SAK and RGD-SAK are shown. The lys35 has been substituted with Arg to constitute a RGD motif and sequence analysis confirmed the successful mutagenesis. (B) Coomassie blue-stained 15% SDS-PAGE showing expression of RGD-SAK. Lane M, protein marker; lanes 1–6, induced for 0, 0.5, 1, 1.5, 2, 2.5 h. (C) Purification of recombinant protein RGD-SAK. Lane M, protein marker; lane 1, cytosol fractions; lane 2, purification after SP-Sepharose; lane 3, purification after Sephadex G-50; lane 4, purification after Q-Sepharose. (D) Western blot analysis with polyclonal anti-SAK antibodies.
Mentions: Using PCR-mediated site-directed mutagenesis, the SAK variant RGD-SAK was constructed with Arg in place of K35, and the desired mutant was identified by DNA sequencing (Fig. 1A). After temperature induction, RGD-SAK was over-expressed due to the temperature inducible pL and pR promoter in the expression vector pLY-4. SDS-PAGE showed that the target protein was expressed after 0.5 h of induction and reached peak after 2.5 h (Fig. 1B). The protein was isolated by homogenization and purified by sequential chromatography through SP-Sepharose, Sephadex G-50, and Q-Sepharose (Fig. 1C). High purity protein was obtained (over 98% by densitometric scanning). Western blot (Fig. 1D) revealed that the RGD-SAK protein had a similar antigen binding capacity to SAK. Using known concentrations of pure SAK as the standard, the yield per liter culture of purified RGD-SAK was estimated to be 0.3 g and more than 60% was recovered in purification.

Bottom Line: Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK.Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Dong' an Road 130#, Fudan university, Shanghai, 200032, PR China. chenhongshan999@sohu.com

ABSTRACT

Background: Although staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK).

Results: By using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.

Conclusion: RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.

Show MeSH
Related in: MedlinePlus