In vitro characterization of a multifunctional staphylokinase variant with reduced reocclusion, produced from salt inducible E. coli GJ1158

Abstract

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T(50%) and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy.

Figures

Figure 1

Cloning of “SAK- RGD- Hirulog” (SRH) and SAK gene into pRSET-A. (a) Amplification of mature SAK and synthetic gene, lane 1: amplification of mature SAK of 420 bp; lane 2 : 100 bp DNA ladder; lane 3: Mutual amplification of synthetic gene of 123 bp. (b) Restriction digestion analyses of SRH, mature SAK and RGD-Hirulog, Lane 1: Double digestion of recombinant plasmid pRSET-A-SRH. The SRH gene of size 543 bp was pop out; Lane 2: Double digestion of recombinant plasmid pRSET-A-SAK. The SAK gene of size 420 bp was pop out; Lane 3: Double digestion of recombinant plasmid pRSET-A-RGD-Hirulog. Synthetc gene of 123 bp was pop out; Lane 4: Triple digestion of recombinant plasmid pRSET-A-SRH. The SAK (420 bp) and synthetic gene “RGD-Hirulog (123 bp)” were pop out; Lane 5: Synthetic gene “RGD-Hirulog” of 123 bp; Lane 6: 100 bp DNA ladder. (c) PCR confirmation of pRSET-A-SAK. Lane 1: 100 bp DNA ladder; Lane 2: Amplification of mature SAK with gene specific primers; Lane 3: Amplification of mSAK with vector specific forward primers T7U and gene specific reverse primers; Lane 4: amplification of mature SAK with gene specific forward primers and vector specific reverse primes T7T. (d) PCR confirmation of pRSET-A-RGD-Hirulog. Lane 1: Mutual amplification of synthetic gene RGD-Hirulog; Lane 2: 100 bp DNA ladder; Lane 3: Amplification of RGD-Hirulog gene with vector specific forward primers T7U and gene specific reverse primers; Lane 4: Amplification of RGD-Hirulog gene with gene specific forward primer and vector specific reverse primer T7.T; Lane 5: Amplification of RGD-Hirulog gene with vector specific primers T7U and T7U.

Figure 2

Outline of the pRSET-A-SRH plasmid (not to scale) indicating the location of SRH sequence insert. The amplified fragment of SAK and synthetic gene were inserted into the BamHI and HindIII sites of pRSET-A expression vector by 3-way ligation reaction for the construction of plasmid pRSET-A-SRH.

Figure 3

Expression and purification of SRH from E. coli GJ1158. Expression and purification analysis SRH under different concentrations of sodium chloride on 15% SDS-PAGE stained with coomassie brilliant blue R-250. Lane 1: SRH induced with 500 mM sodium chloride; Lane 2: SRH induced with 300 mM sodium chloride; Lane 3: Protein marker (14.3, 20.1, 29, 43, 66 97.4 kDa); Lane 4: Ist wash; Lane 5: IInd wash; Lane 4, 5, and 6: purified protein eluates.

Figure 4

Comparison of fibrinolytic activity of SRH and SAK. The fibrinolytic activity was monitored by incubating the fibrin plate containing different concentrations of SRH or SAK at 37°C for 6 h. (a) Results are representative of three independent experiments. The wells A1, A2, and A3 loaded with recombinant SAK while B1, B2, and B3 contain SRH. The well S represents the positive control with 2000 IU of streptokinase while N as a negative control with HBS. 20 μL samples of different concentrations (0.1, 0.2, and 0.4 mg/mL) were loaded into the wells of fibrin plate. The lytic areas are shown as clear zone. (b) Fibrinolytic regression curve of SRH and SAK. The areas of clear zones were plotted over the various concentration proteins. The data shown are mean of three independent experiments.

Figure 5

(a) Time course activation of plasminogen by SRH and SAK. The plasminogen activation activities of SRH and SAK were determined as a function of generated plasmin. Plasminogen (5 μM) and SRH or SAK (10 nM) were incubated at 37°C. At different time point, aliquots were withdrawn and assayed for plasmin activity with Spectrozyme PL (0.3 mM). Data represent a mean ± S.D. of three independent experiments performed under same condition. (b) Antihrombin activity of recombinant protein SRH and SAK. The Antithrombin activity was expressed as inhibition of amidolytic activity of thrombin. Thrombin (200 nM) activity was estimated with Spectrozyme TH (0.3 mM) at the final concentration of 150 μM of SRH or SAK. Data represent a mean ± S.D. of three independent experiments performed under same condition. There are significant statistical differences of antithrombin activity between SRH and SAK (P < 0.05).

Figure 6

Time course of turbidimetric fibrin clot and platelet-rich clot lysis assay. Fibrin clot or platelet-rich clots were incubated with HBS or with recombinant protein SRH or SAK (100 nM) in a solution containing 1.5 μM plasminogen. The decrease in A405 with time was used to calculate the relative clot turbidity at different time point. The experiments were repeated for three times and data represent the means ± S.D. (a) Platelet-rich clot lysis assay: T50% of SAK and T90% of SRH were found 226.98 ± 4.62 min and 76.54 ± 4.74 min, respectively, at the final concentration of 100 nM. Control clots treated with HBS only showed that readings were stable throughout the incubation period. (b) Fibrin clot lysis assay: The T50% of SAK (186.70 ± 2.76 min) and T50% of SRH (191.46 ± 6.65 min) were not significantly different at the final concentration of 100 nM.

Figure 7

The effect of SRH and SAK on inhibition of ADP-induced platelet aggregation. (a) The amplitude-impedance aggregation results are displayed as ohms (Ω) at six minute. The SAK had negligible effect on inhibition platelet aggregation compared to SRH and did not increase with dose. Data represents a means ± SD of 3 independent experiments, which were performed in duplicate. (b) Whole blood aggregometer was used for antiplatelet aggregation study of SRH or SAK in whole blood. Data represent a means ± SD of 3 independent experiments, which were performed in duplicate. Results are expressed as a percentage inhibition in relation to platelets incubated with no recombinant proteins taken as 100%. There are significant statistical difference between the inhibitory effect of SRH and SAK for each concentration (P < 0.05).