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Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3
Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3
Journal of Microbiology and Biotechnology. 2015. Sep, 25(9): 1449-1459
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : March 04, 2015
  • Accepted : May 23, 2015
  • Published : September 28, 2015
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About the Authors
Guangyan Cheng
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, P.R. China
Liying He
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P.R. China
Zhibin Sun
School of Life Science, Nanjing Agriculture University, Nanjing 210095, P.R. China
Zhongli Cui
School of Life Science, Nanjing Agriculture University, Nanjing 210095, P.R. China
Yingxiang Du
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P.R. China
du_yingxiang@126.com
Yi Kong
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, P.R. China
du_yingxiang@126.com

Abstract
A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg 2+ and Ca 2+ and completely inhibited by Cu 2+ , but slightly enhanced by Fe 2+ . According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.
Keywords
Introduction
Cardiovascular diseases (CVDs), including coronary heart disease, cerebrovascular disease, hypertension, peripheral artery disease, rheumatic heart disease, congenital heart disease, and heart failure, are the major cause of death throughout the world [41] . According to data from the World Health Organization (WHO), about 17.5 million people died from CVDs in 2005, representing 30% of all global deaths. The WHO has also predicted that the situation will continue to worsen over time. By 2030, about 23.6 million people will die from CVDs every year [23] . Accumulation of fibrin clots in blood vessels often increases thrombosis, and is one of the main causes of CVDs. Fibrin is formed from fibrinogen by thrombin (E.C. 3.4.21.5) and can be lysed by plasmin (E.C. 3.4.21.7), which is generated from plasminogen by plasminogen activator.
Treatment of CVDs originating from thrombosis is largely based on hydrolyzing fibrin using fibrinolytic agents. The major thrombolytic agents are classified into two types based on their mechanisms. One is the plasminogen activators, such as urokinase [13] , tissue-type plasminogen activator [9] , and streptokinase [52] , which activate plasminogen to plasmin, and then eventually hydrolyze fibrin. The other is plasmin-like proteins that directly degrade fibrin. The typical thrombolytic agents used for therapeutic purposes are plasminogen activators. However, all these enzymes have drawbacks, such as an allergenic nature, high cost, low fibrin specificity, short half-life, induction of gastrointestinal bleeding, and resistance to repercussion, which limit their clinical use [20] . Direct thrombolytic agents such as plasmin that degrade fibrin clots have shown encouraging biochemical and preclinical results [40 , 49] . However, plasmin is rapidly inactivated by α2-antiplasmin [8] . Therefore, the search for more economical and safer thrombolytic agents from diverse sources is ongoing.
In recent years, many thrombolytic agents have been identified and characterized from different sources, including fermented food products such as Japanese natto [50] , Korean chunkook-jang [29] , and Chinese douchi [54] , foodgrade microorganisms [59 , 10] , insects [1] , polychaetes [11] , earthworms [54 , 55] , and snake venom [12] . Thrombolytic agents from microorganisms have increasingly attracted medical interest because of their broad biochemical diversity, feasibility of mass culture, and ease of genetic manipulation. Hence, many fibrinolytic enzymes have been isolated from a variety of microorganisms, including bacteria, fungi, and algae. Among them, only a few have been discovered from Streptomyces , including a 23 kDa plasminogen activator and plasmin-like fibrinolytic serine protease (SOT) from Streptomyces omiyaensis [53] , an 18 kDa chymotrypsin-like serine metalloprotease with fibrinolytic activity (FES624) from Streptomyces sp. CS684 [48] , a 35 kDa chymotrypsin-like serine peptidase with fibrinolytic activity from Streptomyces megaspores SD5 [4] , and a 30 kDa plasmin-like fibrinolytic enzyme (SW-1) from Streptomyces sp. Y405 [57] .
In this study, we describe a potent thrombolytic agent from Streptomyces . We describe the purification, biochemical properties, and fibrinolytic activity of a fibrinolytic protease produced by Streptomyces sp. P3.
Materials and Methods
- Materials
Bovine fibrinogen, bovine thrombin, human plasminogen, azocasein, phenyl-methylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), and ethylene glycolbis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) were purchased from Sigma-Aldrich (MO, USA). DEAE-Sepharose FF and Sephadex G-50 were obtained from GE Health-care Life Sciences (USA). Urokinase was purchased from the National Institutes for Food and Drug Control (Beijing, China). LA Taq DNA polymerase, 2× GC buffer I, dNTP mixture, DL5000 Marker, pMD19-T vector, T4 ligase, and LMW standard protein markers were purchased from Takara Co., Ltd (Dalian, China). Other reagents and chemicals were of analytical grade and commercially available.
- Strain Isolation and Screening
Streptomyces strains were isolated from air-dried soils collected from different areas of China according to an intensive method developed by Hayakawa and Nonomura [22] . The strains were cultivated in improved HV-agar plates at 28℃ for 5 days. Single colonies were inoculated into 20 ml of HV medium and cultivated at 30℃, 150 rpm for 48 h, and the fermentation broth was assayed for proteolytic enzyme activity. Among the colonies, those with proteolytic activity were then screened using a fibrin plate. Single colonies capable of producing a clear zone in the fibrin plate were picked and cultivated in improved HV medium. Using this method, Streptomyces sp. P3 was proved to be potent with a highly fibrinolytic enzyme. Streptomyces sp. P3 was cultured in an improved HV medium and cultivation was carried out for 7 days on a shaker set at 30℃ and 180 rpm. The improved HV medium contained (g/l) soluble starch, 8; KNO 3 , 0.5; KCl, 1.7; MgSO 4 , 0.5; Na 2 HPO 4 , 1; CaCO 3 , 0.02; and FeSO 4 , 0.01.
- Determination of Protein Concentration
Protein concentration was determined by the method of Lowry [36] using bovine serum albumin as a standard. The concentration of protein was determined by measuring the absorbance at 750 nm.
- Assay of Enzyme Activity
Protease activity was determined using azocasein as the substrate according to a previously described method [11] with some modifications. A total of 100 μl of the purified enzyme was mixed with 400 μl of 2 mg/ml azocasein in 20 mM sodium phosphate buffer (pH 7.5). Following incubation at 37℃ for 30 min, 500 μl of ice-cold 10% (w/v) trichloroacetic acid was added to the mixture and vortexed. The mixture was placed at 4℃ for 10 min and centrifuged at 10,000 × g for 10 min. The absorbance of the supernatant was measured at 340 nm. One unit of protease activity was defined as the amount of enzyme causing an increase in absorbance of 0.001 at 340 nm.
- Fibrinolytic Activity Assay
Fibrinolytic activity was determined by both the modified plasminogen-free fibrin plate and the plasminogen-rich fibrin plate methods as described by Astrup and Müllertz [2] with slight modifications. A plasminogen-free fibrin plate was obtained by heating a plasminogen-rich fibrin plate at 85℃ for 30 min. Plasminogen-rich fibrin plates were prepared by adding a solution composed of 4 mg/ml fibrinogen in 20 mM sodium phosphate buffer (pH 7.5), 1.0% agarose, 0.5 U/ml thrombin, and 0.2 ml of plasminogen (50 U/ml) to a petri dish. The clot was allowed to set for 30 min at room temperature and then 10 μl of the enzyme sample was added into a well (3.0 mm diameter) made in the gel. The plates were incubated at 37℃ for 18 h. Fibrinolytic activity was calculated by measuring the diameter of the transparent zone around the well [17] . Urokinase was used as the positive control.
- Purification of Fibrinolytic Enzyme
Streptomyces sp. P3 was cultured for 7 days, centrifuged at 12,000 × g for 30 min at 4℃, and ammonium sulfate at 60% saturation was added to the supernatant to precipitate proteins. Precipitated proteins were recovered by centrifugation at 12,000 × g for 1 h at 4℃, dialyzed against 20 mM sodium phosphate buffer (pH 7.5), and desalted using a dialysis membrane with a 3,500 Da cutoff. The crude enzyme solution was purified using a DEAE-Sepharose FF column (1.0 × 20 cm) pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.5). The bound proteins were eluted using a gradient of 20 mM sodium phosphate buffer (pH 7.5) containing 0–0.5 M NaCl at a flow rate of 0.5 ml/min. The major active fraction was pooled, concentrated with a YM10 ultra filtration membrane (Millipore Corporation, USA), and then subjected to gel filtration using a Sephadex G-50 column (2.6 × 100 cm) previously equilibrated with 20 mM sodium phosphate buffer (pH 7.5). Proteins were eluted in the same buffer at a flow rate of 0.3 ml/min. The active fraction was desalted and analyzed by gel electrophoresis. For all purification steps, the eluates were monitored by spectrophotometry at 280 nm. The activity of the enzyme was estimated with the azocasein method.
The molecular mass of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described by Laemmli [31] using a 5% (w/v) stacking gel and 12% (w/v) polyacrylamide resolving gel. A low-molecularweight marker was used as the reference protein.
- Effect of Temperature on Enzyme Activity and Stability
To determine the optimal temperature, the enzyme assay was carried out with azocasein at various temperatures (4–70℃) and pH 7.5. The thermal stability of the enzyme was evaluated by incubating the enzyme sample at various temperatures for 15–90 min at pH 7.5 and then the residual activities were measured. The initial enzyme activity (prior to exposing the enzyme to the various temperatures) was considered to be 100%.
- Influence of pH on Enzyme Activity and Stability
To determine the optimum pH, the assay was carried out with azocasein at different pH values (pH 3.0–12.0) using standard buffers (100 mM): citric acid-sodium phosphate (pH 3.0–7.0), Tris-HCl (pH 7.5–9.0), and NaHCO 3 (pH 10.0–12.0). The stable pH range was obtained by measuring the residual activities after incubating the enzyme for 24 h at 4℃ at each pH (100 mM) over the ranges specified. The residual enzyme activities were calculated by comparison with the initial activity, which was set at 100%.
- Influence of Enzyme Inhibitors and Metal Ions
The enzyme was incubated with different metal ions and inhibitors for 30 min at room temperature and the residual activity was measured. The effects of metal ions such as CaCl 2 , MgSO 4 , CuSO 4 , and FeSO 4 and inhibitors such a s EDTA, EGTA, and PMSF were measured. The enzyme activity measured without metal ions and inhibitors was considered as 100%.
- Analysis of the Amino Acid Sequence by LC-MS/MS
The purified enzyme was analyzed by 12% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue R250. The purified enzyme was excised from the SDS-PAGE gel as a single band and was analyzed by LC-MS/MS (Bo-Yuan Biological Technology Co. Ltd., Shanghai, China). The obtained amino acid sequence was submitted to the National Center for Biotechnology Information (NCBI) database. Homology with the amino acid sequence of FSP3 was determined by searching through nonredundant protein sequences and databases with the NCBI BLAST program.
- Cloning and Sequencing of the FSP3 Gene
To clone the gene encoding FSP3, Streptomyces sp. P3 genomic DNA was extracted from a single colony to be used as a template for the polymerase chain reaction (PCR) by the quick and easy KOH-EDTA lysis method [51] . Several pairs of primers were designed to amplify the FSP3 gene based on the fragments determined by analysis of LC-MS/MS results ( Table 1 ). The full length of the FSP3 gene was amplified by PCR. The reaction mixtures were prepared in a total volume of 25 μl containing approximately 50 ng of genomic DNA, 2.5 mM MgCl 2 , 3.2 mM dNTP, 2.5 U of Taq polymerase, and 0.6 μM of each primer. PCR amplification was carried out in a DNA thermocycler (Perkin-Elmer) under the following steps: 1 cycle at 95℃ for 5 min, 30 cycles of denaturation at 94℃ for 0.5 min, annealing to 55℃ for 0.5 min, and extension at 72℃ for 1.5 min, and an additional extension at 72℃ for 5 min and 10℃ for 10 min. Nucleotide sequences were determined by Genscript Technologies Co. (Nanjing, China). Analysis and comparison of amino acid or nucleotide sequences were performed using BLAST provided by the NCBI website ( http://www.ncbi.nlm.nih.gov/ ).
Primers for PCR amplication used in this study.
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Primers for PCR amplication used in this study.
The bacterial strains and plasmids used in this work are listed in Table 2 . Unless otherwise stated, cells were grown at 37℃ in Luria-Bertani (LB) broth with shaking. When required, media were supplemented with 100 μg/ml ampicillin (Amp) for Escherichia coli .
Strains and plasmids used in this study.
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Strains and plasmids used in this study.
- Tail-Bleeding Time
The tail-bleeding time, used to assess hemostasis, was carried out according to described methods [19 , 59] with slight changes. Mice were anesthetized and placed on a 37℃ heating pad. The tail was transected 3 mm from the tip and immediately immersed in normal saline (37℃), and the bleeding time was recorded. If the bleeding did not stop within 30 min, the bleeding time was defined as >1,800 sec. Vehicle, urokinase (300 U), or FSP3 was administered by intravenous injection with six mice per group.
Results and Discussion
- Enzyme Purification
Purification of FSP3 was carried out in three successive steps as shown in Table 3 . The crude extract contained 422.4 mg protein; maximum activity was obtained by precipitation of 60% saturation of ammonium sulfate. The major fraction with fibrinolytic activity was applied to the DEAE-Sepharose FF column, which generated one single peak showing fibrinolytic activity in the eluate ( Fig. 1 A). The major active fraction was pooled, concentrated, and further purified using gel filtration via a Sephadex G-50 column, and a single peak with a high specific activity was acquired ( Fig. 1 B). Overall, 26-fold purification and recovery of 28.3% activity (yield) were obtained after completion of the purification steps. The specific activity of the final enzyme preparation was 11,792.6 U/mg protein. The purified enzyme was designated as Streptomyces sp. P3 fibrinolytic protease (FSP3). SDS-PAGE of the purified enzyme was performed to verify enzyme purity and determine the molecular mass. The protein migrated as a single band and the molecular mass was estimated to be 44 kDa ( Fig. 2 ). The molecular mass of the reported microbial fibrinolytic enzymes is in the range of 22.7-63.3 kDa [37 , 46 , 60] . The molecular mass of the purified enzyme from Streptomyces sp. P3 was the same as that for the fibrinolytic enzyme from B. subtilis KCK-7 (44 kDa) [43] , and was similar to that of the fibrinolytic enzyme from Bacillus sp. KDO-13 (45 kDa) [32] and the fibrinolytic enzyme from Bacillus sp. KA38 (41 kDa) [26] . However, the fibrinolytic enzyme obtained in this study was smaller than that from C. militaris (52 kDa) [27] and was much larger than the subtilisin DFE from B. amyloliquefaciens DC-4 (28 kDa) [45] , A. mellea (21 kDa) [34] , and P. eryngii (14 kDa) [3] .
Purification of the enzyme.
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Purification of the enzyme.
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Elution profile of the enzyme from ion exchange with DEAE-Sepharose column (A) and gel filtration with Sephadex G-50 column (B). Protein concentration (□) and enzyme activity (●) of each fraction were measured at 280 and 340 nm, respectively. Enzyme activity was expressed in terms of U/ml.
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Electrophoretic analysis of the purified enzyme carried out in a 12% (w/v) polyacrylamide slab gel according to the method of Laemmli. The gel was stained with Coomassie Brilliant Blue R-250 and destained with methanol/glacial acetic acid/distilled water (1:1:8, by vol.). 1, Protein marker; 2, purified enzyme.
- Fibrinolytic Activity Assay
The fibrinolytic activity of FSP3 was estimated using the fibrin plate method. After incubation at 37℃, FSP3 was observed to form a larger zone around the well than treatment with an equal amount of urokinase ( Fig. 3 A). As the area of the clear zone is directly proportional to the activity, it was concluded that FSP3 has stronger fibrinolytic activity than that of urokinase. Unlike urokinase, the purified enzyme formed a clear zone in the plasminogenfree fibrin plate, which indicated that the purified enzyme has plasmin-like activity that can degrade the fibrin clot by direct fibrinolysis. The dimension of the clear zone of the purified enzyme increased in the presence of plasminogen, suggesting that the purified enzyme also has plasminogen activator-like activity ( Fig. 3 ). In the plasminogen-rich fibrin plate, the fibrinolytic activity of the purified enzyme was about 1.2-fold higher than that of urokinase. In the plasminogen-free fibrin plate, in the presence of plasminogen, the fibrinolytic activity of the purified enzyme was about 1.4-fold higher than that of urokinase. Taken together, the results suggest that the fibrinolytic activity of the purified enzyme is comparable to that of urokinase. The size of the clear hollow zone changed obviously in the presence of plasminogen, suggesting that FSP3 is bifunctional in nature, capable of hydrolyzing the fibrin directly and as a plasminogen activator. FSP3 is a plasmin-like protease and a plasminogen activator consistent with codiase, a bifunctional fibrinolytic enzyme from Codium fragile [5] , SOT from Streptomyces omiyaensis [53] , and NJP from the polychaete Neanthes japonica [58] . The typical thrombolytic agents for therapeutic application are plasminogen activators such as urokinase, tissue-type plasminogen activator, and streptokinase. Its bifunctionality gives FSP3 an advantage over the clinically used plasminogen activators.
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Assay of fibrinolytic activity of the purified enzyme on a plasminogen-rich fibrin plate (A) and a plasminogen-free fibrin plate (B), heated at 80℃ for 30 min. 1, Purified enzyme (100 U/ml); 2, purified enzyme (100 U/ml) and plasminogen (10 U/ml); 3, urokinase (100 U/ml) and plasminogen (10 U/ml); 4, urokinase (100 U/ml).
- Effects of pH and Temperature
The activity and stability of FSP3 were highly affected by temperature and pH. A comparison of the pH and thermal characteristics of FSP3 with those of related enzymes is presented in Table 4 . The influence of pH on the activity of FSP3 was determined using buffers at various pH values ranging from 3.0 to 11.0. The optimal pH for proteolytic activity was determined to be 6.5 ( Fig. 4 C), which is similar to the optimum observed for AprE86-1 (6.0–7.0) [33] , AprE3-17 (6.0) [24] , and FVP-1 (6.0) [44] , but lower than those of CDP (9.0) [41] , subtilisin DFE (9.0) [46] , and subtilisin FS33 (8.0) [54] . As shown in Fig. 4 D, the enzyme was stable at pH 6.0–8.0, and the activity was greatly decreased below pH 6.0 and over 8.0, exhibiting a pH stability comparably wider than that of B. subtilis [28] . These results suggest that the enzyme is active over a very narrow range of pH values, which indicates that FSP3 may be suitable for use in the human in vivo environment.
Comparison of the characteristics of FSP3 with those of other fibrinolytic serine proteases.
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Comparison of the characteristics of FSP3 with those of other fibrinolytic serine proteases.
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Effects of temperature and pH on enzyme activity and stability. (A) Temperature optimum of the enzyme was determined by measuring the activity at various temperatures for 30 min. (B) Thermal stability of the enzyme was assessed by measuring the residual activity after the enzyme was incubated at 37℃ (●), 40℃ (■), and 50℃ (▲) for 15-90 min. (C) pH optimum was determined by measuring enzyme activity in different pH buffers: 100 mM citric acid-sodium phosphate (pH 3.0-7.0), Tris-HCl (pH 7.5-9.0), and NaHCO3 (pH 10.0-12.0). (D) pH stability was determined by measuring the residual activity after the enzyme samples were incubated in different buffers at 4℃ for 24 h.
FSP3 showed maximal activity at 50℃ and remained stable at or below 40℃ ( Figs. 4 A and 4 B). The optimal temperature for FSP3 coincided with that for FP28 [47] and Bacillokinase II [61] and was lower than that reported for AJ (85℃) [6] , but higher than that reported for subtilisin DFE (48℃) [46] , N-V protease (45℃) [62] , CSP(40℃) [35] , and MEF (30℃) [18] . As shown in Fig. 4 B, enzyme activity was very stable below 40℃, which corresponds to the temperature in the human body.
- Effects of Protease Inhibitors and Metal Ions
The influence of different metal ions on enzyme activity was detected by measurement of residual enzyme activity after incubation of the enzyme with 5 mM of each metal ion and protease inhibitors for 1h at 37℃. The effects of various inhibitors and metal ions are summarized in Table 5 . The enzyme was completely inhibited by 5 mM PMSF, which is a known serine protease inhibitor. However, 5 mM EDTA and 5 mM EGTA, metalloprotease inhibitors, did not obviously inhibit enzyme activity. This result suggests that the FSP3 enzyme is a serine protease, similar to a fibrinolytic enzyme from the newly isolated marine bacterium Bacillus subtilis ICTF-1 [38] and NJP [58] . Most reported fibrinolytic enzymes are serine proteases, such as EFEa–g [56] , nattokinase [14] , and the fibrinolytic enzyme from Bacillus lichenifomis KJ-31 [21] . Furthermore, metal ions exhibited a different influence on FSP3 activity. Cu 2+ completely but Ca 2+ and Mg 2+ partially inhibited the activity of FSP3, whereas Fe 2+ promoted it ( Table 5 ).
Effect of metal ions and protease inhibitors.
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Effect of metal ions and protease inhibitors.
- LC-MS/MS Analysis of FSP3
The purified protein from the band on SDS-PAGE was analyzed by LC-MS/MS. The partial amino acid sequences of the three peptides obtained were MARIGDGGDLLK, SNILLLGPTGSGKTLLAQTLAR, and AGAKGIGFGATIRSK. These sequences were compared with the other sequences available in the NCBI protein database using BLAST. From this homology search, we presumed the possible amino acid sequence encoding the mature peptide of FSP3. The result of LC-MS/MS and the possible sequence of FPS3 are shown in Fig. 5 . The BLAST search indicated that FSP3 exhibits high homology with the sequences of an ATPdependent Clp protease from Rhodopseudomonas palustris DX-1.
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LC-MS/MS analysis and peptide sequence matches of FSP3. LC-MS/MS fragmentation sequencing spectra of fragment 2(A) and fragment 3(B). Observed ions of each type in each case are shown in bold typeface. (C) Possible sequence of FSP3. Matched peptides are shown in bold typeface.
- Cloning, Sequencing, and Analysis of the FSP3 Gene
A conserved region of 306 bp was obtained by the primers F(337) and R(643) designed based on the identified peptide sequence of LC-MS/MS. However, we could not obtain the whole sequence of FSP3 through this method. Therefore, the tail-PCR method was attempted in order to clone the full-length gene of FSP3, but it too did not succeed. Then, we designed several primers from the nucleotide sequence corresponding to the FSP3 mature peptide and the 306 bp conserved region to amplify the full-length sequence by arbitrarily primed PCR. Finally, the full DNA sequence of FSP3 was amplified by the forward primer F(1) and reverse primer R(1286). Sequencing of the amplified fragment revealed that there is 91% similarity between the mature peptides of FSP3 and the ATPdependent Clp protease, and 83% similarity with NK and 89% similarity with the fibrinolytic enzyme from Bacillus sp. ZLW-3. The mature FSP3 protein is composed of 428 amino acids. This putative mature peptide has the same molecular mass as that determined by SDS-PAGE for the previously purified FSP3 (~44 kDa), consistent with the cloned DNA fragment corresponding to the FSP3 gene.
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Cloned DNA fragment encoding FSP3. M: DL5000 marker.
Furthermore, in our study, the gene FSP3 was recombined with the plasmids pET29a(+), pP43NMK, and pMA09, and then the recombinant plasmid pET29a(+)-FSP3 was transformed into E. coli BL21 [16] , and pP43NMK-FSP3 and pMA09-FSP3 were transformed into Bacillus subtilis WB800 [15] . The transformed cells were grown in LB medium to express FSP3 in the host bacteria, but no protease activity was detected, perhaps because this protease was toxic to the expression hosts, or because the serine codon, which is part of the catalytic triad of serine proteases, is a rare codon in Bacillus subtilis WB800. Alternatively, the lack of expression could have been caused by low affinity between the host bacteria and Streptomyces sp. P3. Thus, further research into the expression of the protease gene using a Streptomyces -derived system is required.
- Influence of FSP3 on Bleeding
To evaluate the influence of bleeding risk caused by FSP3, a tail-bleeding assay was performed in a mice model. FSP3 (300 U) with a strong fibrinolytic effect did not prolong the bleeding time ( Fig. 7 ), suggesting that its fibrinolytic efficacy would not be complicated by any bleeding side effects. Urokinase, however, noticeably prolonged the bleeding time. In two mice out of six, the bleeding time exceeded 30 min. There is always the possibility that a bleeding complication may arise from treatment with an antithrombotic drug. Nevertheless, FSP3 given at a high fibrinolytic dose did not produce bleeding complications in a mouse tail-bleeding assay. This result may be because FSP3 showed both plasmin-like activity and plasminogen activator activity.
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Effect of FSP3 on bleeding time. Saline, urokinase (300 U), and FSP3 (300 U) were injected intravenously into six mice per group.
In this study, we have purified and characterized a novel protease (FSP3) from Streptomyces sp. P3 that is clearly different from other fibrinolytic enzymes. FSP3 is a serine protease with a much higher fibrinolytic activity than that of urokinase. It can directly degrade fibrin and also act as a plasminogen activator. FSP3 may be a potential candidate for thrombosis prevention and thrombolytic therapy. Further studies of FSP3 in terms of its physiological function and its effectiveness at limiting in vivo thrombolysis are required.
Acknowledgements
This study was supported by the Chinese National Natural Science Foundation (81273375) and the Jiangsu Provincial Qing Lan Project.
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