Running title: Pilot-scale production and purification of staphylokinase-based fusion protein

Pilot-scale production and purification of staphylokinase-based fusion protein over-expressed in Escherichia coli

Genshen ZHONG1, Aiping YU ()1, Bingxing SHI1, Yang LIU2, Chutse WU1,2 1 Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China 2 Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Abstract SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the
N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.

Keywords

staphylokinase, E. coli, purification, endotoxin

Introduction

A new fusion protein, SFH, composed of staphylokinase (SAK) and hirudin (HV), with the coagulant factor Xa (FXa) recognition sequence as the linker peptide, has been designed and expressed in engineered Escherichia coli BL21 (DE3) bacteria. The results from animal experiments suggest that SFH possesses excellent bifunctional activities of anticoagulation and thrombolysis, which solves the dilemma of how to balance anticoagulant activity with hemorrhagic events (Shi et al., 2007). Thus, SFH is a promising candidate for effective and safe therapy for thrombosis in clinics. However, the yield of SFH expressed in E. coli is less than 100 mg/L under lowcell-density cultivation (unpublished data), incapable of meeting the needs of preclinical experiments and potential clinical thrombolysis therapy in the future. Therefore, how to increase the yield of SFH is an urgent problem that needs to be solved. The high-density cultivation of recombinant E. coli resulting in increased volumetric productivity has proven to be a practical approach to acquire high-yield heterogenetic protein in E. coli (Choi et al., 2006; Shiloach and Fass, 2005). The fed-batch growth technique is one of the most effective ways to achieve high cell density, and several fed-batch methods have been developed (Lee et al., 1999). The fed-batch strategy with dissolved oxygen feedback control is a simple and easily-controllable approach for cultivation (Manderson et al., 2006; Yano et al., 1978; Johnston et al., 2002). In this report, we describe a simple and easily-scalable purification process with expanded bed adsorption chromatography (EBAC) and anion-exchange chromatography (IEC) to purify SFH from E. coli lysate. The goal of this
E-mail: wuct@bmi.ac.cn 1

work was to produce adequately pure SFH for animal toxicological and preclinical studies. 2 2.1 Materials and methods Strain and plasmid

The strain E. coli BL21 (DE3) was used as the host strain to express the fusion protein SFH. The construction of the temperature-sensitive expression recombinant plasmid pBV220-SFH was described in our previous report (Shi et al., 2007). 2.2 High-cell-density cultivation The engineered strain E. coli BL21(DE3)/pBV-SFH was stored at 80C in a 30% glycerol solution until use. For cultivation, the stored cell suspension was diluted and cultivated for 20 h at 37C in LB-agar medium containing 50 g/mL ampicillin. A single colony from an agar plate was transferred to 50 mL LB medium containing 100 g/mL ampicillin in a 500 mL Erlenmeyer flask and incubated at 30C for 10 h on a rotary shaker (280 r/min). Thirty mL of the above seed culture was transferred to 1.5 L LB medium containing 100 g/mL ampicillin in a 2 L Erlenmeyer flask, then divided into 15 equal parts and put into a 500 mL Erlenmeyer flask separately and incubated at 30C for 12 h on a rotary shaker (220 r/min). Thereafter, the seed culture was inoculated into a 40 L bioreactor (B. Braun, Germany) in complex medium, and the initial working volume was 20 L. The composition of the complex medium was as follows: KH2PO4, 3.0 g/L; K2HPO4, 6.0 g/L; (NH4)2SO4, 2.0 g/L; yeast extract, 5 g/L (Merck); peptone, 10 g/L (Merck); glucose, 6 g/L; MgSO47H2O, 1.0 g/L; ampicillin, 0.1 g/L; antifoam 0.1 mL/L (Sigma), and trace elements 1.5 mL/L with reference to Riesenberg (Riesenberg et al., 1991). The maximum values of agitation speed and flow rate of aeration were set at 800 r/min and 28.5 L/min, respectively, on which the dissolved oxygen (DO) was set at 100% air saturation. Fermentation was carried out at 30C. The agitation speed was 300 r/min and the flow rate of the aeration was 10 L/min at the start. The pH of the medium was maintained at 7.0 by automatic addition of aqueous ammonia (25% v/v). After the initial glucose in the bioreactor was consumed, the DO was decreased first and then increased dramatically. Feeding was initiated with a supply of the mixture: glucose, 350 g/L; yeast extract, 70 g/L; peptone, 140 g/L; MgSO47H2O, 15 g/L, and the feeding speed was increased stepwise based on the DO fluctuation, which was maintained above 20% air saturation by adjusting the agitation speed, the flow rate of the aeration, and the feeding speed. When the agitation speed and flow rate of the aeration reached the maximum of the setting, adjustment of the feeding speed became the major means to maintain the DO above 20% air saturation. The residual glucose was determined with the Miller method (Miller, 1959) and the final cultivation volume was about 24 L. The culture with 50--60 OD600 (optical density) was induced by a temperature shift from 30C to 42C for another 4 h, and then the E. coli bodies were harvested by centrifugation at 5000 g, 4C for 15 min. The harvested cells were frozen and thawed for three cycles according to the method developed by Johnson (Johnson and Hecht, 1994), and suspended in 4.5 L 20 mM Tris-HCl buffer (pH 8.0) for 4 h. The supernatant was obtained after centrifugation at 10000 g, 4C for 30 min. Another 4.5 L 20 mM Tris-HCl buffer (pH 8.0) was then added to re-suspend the precipitate of the E. coli bodies and centrifuged after the freezing and thawing cycles. The supernatant was obtained again. This protein extraction procedure was repeated four times. Finally, about 22 L periplasmic fraction solution was obtained at one operation on a 40 L cultivation scale. The periplasmic fraction was ultrafiltrated through a hollow fiber column (Millipore) with a cutoff of 100 kDa, and then the clarified supernatant was applied to the following purification steps. The protein concentration was determined with the Lowry method, and the SDS-PAGE was carried out according to Laemmlis method. 2.3 Purification The purification procedures were performed on an KTA Prime or an KTA Purifier system (Amersham Biosciences). The proteins were monitored at 280 nm on a UV detector. All the operations were carried out under endotoxin-free conditions in the whole purification process. 2.3.1 Chromatography by expanded bed adsorption The clarified supernatant was pumped upward on pre-equilibrated STREAMLINE Q expanded in a STREAMLINE 400 column at a ratio of 150 mg cell extract to 1 mL packed column at a flow rate of 300 cm/h. After all of the cell suspension was applied to the column and the upward flow was stopped, the bed was allowed to settle and the flow adaptor was lowered to the surface of the packed bed. After washing with 20 mM Tris-HCl buffer (pH 8.0) in a downward mode with
2

one column volume at a rate of 60 cm/h, the protein was eluted with a 0--1 M NaCl gradient. The target peak was collected for the next step of purification. 2.3.2 Ion-exchange chromatography After being diluted twice with 20 mM Tris-HCl buffer (pH 8.0), the desired SFH collected from Streamline Q was loaded onto a Source 15Q (Amersham) column (XK50, 20 cm 5 cm) pre-equilibrated with 20 mM Tris-HCl (pH 8.0) at a flow rate of 45 cm/h. After sample loading, the protein was eluted using a gradient from 0--0.6 M NaCl. The fractions containing SFH were pooled and could be desalted and concentrated to 10 mg/mL using a flat-plate membrane filter (Millipore) with a cutoff of 8 kDa. Thereafter, a stabilizer [mannitol/sucrose, 3%/1% (w/v)] was added into the concentrated fraction and was transferred in 1.0 mL aliquots to 3 mL glass vials, which could be lyophilized for further use. 2.4 Multi-charged ESI-MS analysis For the ESI-MS studies, the protein was dialyzed against 50 mM phosphate buffer, pH 7.4, and then diluted to 2 mg/mL. An accurate mass was obtained at the GENOPOLE facilities at The National Institute for the Control of Pharmaceutical and Biological Products in Beijing, China. The programs BiomultiView and the PeptideMap were used to determine the molecular mass. 2.5 Measurement of fibrinolytic activity The fibrinolytic activity (Saksela, 1981) of SFH was determined according to the proteolytic activity monitored on a 5% agar plate containing 1 NIHU/mL of human thrombin (Sigma), 10 g/mL of human plasminogen (Roche) and 0.8 mg/mL of fibrinogen (Chinese Medical Academy) at pH 7.5. Ten L samples with serial dilutions of SFH and SAK were added to the pre-cast wells. The plates were incubated at 37C for 4 h and the fibrinolytic activities were expressed as the diameter of the clear zones around the wells. The standard SAK with assigned fibrinolytic activity 100 000 IU/mg was purchased from the State Food and Drug Administration of China. 2.6 Endotoxin determination by the Limulus amebocyte lysate method The endotoxin level was determined with the Limulus amebocyte lysate (LAL) test using an end-point method as described in our previous report (Shi et al., 2006). 3. 3.1 Results Temperature-sensitive expression of the SFH in high-cell-density culture of the recombinant E. coli BL21(DE3)

Before the pilot-scale cultivation, the cultivation conditions including the E. coli host strain, medium composition, pH, cultivation time and induction time were all optimized at the bench scale in a 5 L bioreactor under high-cell-density cultivation (Zhong et al., 2006). Under the cultivation conditions optimized at the bench scale, the engineered E. coli bacteria were cultivated in a 40 L bioreactor to achieve a high cell density. After cultivation for about 4 h, the DO began to decrease dramatically and later rose sharply (Fig.1A). The pH also increased obviously because of the excretion of ammonium ions (Lee, 1996) (data not shown). All these indicated that the carbon source was insufficient when the feeding began. The agitation speed, the flow rate of aeration, and the feeding speed were adjusted to maintain the DO above 20% air saturation. After cultivation for 16 h (DCW = 27 g/L), the temperature was shifted from 30C to 42C to induce the expression of SFH. After an additional 4 h cultivation, the DCW was increased to about 41 g/L, and the expression level of SFH was increased with induction time (Fig.1B). After harvesting by centrifugation, the periplasmic fraction was obtained as described in Materials and Methods. The total protein was about 4.5 g/L, and the percentage of SFH was more than 25% of the total protein (Fig.1B), i.e. the SFH yield was approximately 1.1 g/L. This pilot-scale cultivation process was repeated for more than 3 times in a 40 L bioreactor, and the results were of good reproducibility (Table 1).

Fig. 1
3

Table 1 Pilot-scale purification of the recombinant SFH in a 40 L bioreactor

step

total volume/mL 22033356 21233238 10473446 6207340

total proteina/g 109.46.3 92.82.0 24.82.2 14.71.6

total SFH/106 1145118.9 109088.0 866140.3 65259.1

specific activityb /IUmg

-1

recovery yield/% 100 95.32.2 75.54.5 57.03.0

endotoxin level/EUmL-1 100000 8000 2113 c 0.5 d

E. coli lysate filtered broth streamline Q source 15Q

a b c d

10453502 11740706 348242593 443011103

The results represent meanSD of 3 preparations. : Determined by the Lowry method using bovine serum albumin as a standard; : Determined by the fibrinolytic activity assay using a standard SAK with an assigned activity of 100000 IUmg-1; : Below 16 EUmL-1 in 2 preparations and below 32 EUmL-1 in the third preparation; : All 3 preparations were below 0.5 EUmL-1. Purification and ESI-MS analysis of the SFH

3.2

A two-step procedure consisting of the EBAC and IEC was used to purify the SFH. The EBAC is a primary recovery operation allowing the adsorption of proteins directly from the un-clarified feedstock. However, it has been reported that the interaction between the cells and the adsorbent in the expanded bed has some potential negative influence on protein separation (Lin et al., 2004; Fernandez et al., 2000). Therefore, after the extraction procedures, the periplasmic fraction solution of SFH was first treated with the hollow fiber column with the 100 kDa cutoff for fast removal of most of the cells and the cell debris, and then the clarified sample was applied onto the EBAC. In the EBAC purification, SFH elution by a gradient of 0--1 M NaCl was done at about 20% NaCl concentration (Fig.2A). As seen in the electrophoresis pattern in Figure 2C, the purity of the eluate of interest pooled from the EBAC was more than 80%. The major contaminants were flow-through and removed in this step. Compared to the feedstock, the endotoxin content also decreased dramatically to less than 2113 EU/mL (Table 1), which indicated that the EBAC was an effective way to remove the endotoxin. The recovery yield of SFH in this step was estimated to be about 76% based on the fibrinolytic activity assay. After EBAC separation, the solution collected from the EBAC was diluted and applied onto the IEC. The chromatographic profiles of the target protein separated by the IEC are shown in Figure 2B. It was observed that the target peak SFH was present within the range of 0.12 to 0.20 M NaCl gradients identified by the SDS-PAGE assay (Fig.2C). The recovery yield of SFH on the IEC was about 75% and the residual endotoxin content was less than 0.5 EU/mL after the IEC separation, and was valid for further preclinical or clinical studies. The total yield of the final purified SFH was about 600 mg/L (Table 1). A typical purification process resulted in over 99.0% purity of the SFH analyzed by RP-HPLC (data not shown).

Fig. 2 The accurate multi-charged ESI-MS was used to determine the molecular weight (MW) of SFH. The theoretical MW of SFH was 22999.82 Da based on the amino acid composition, and the MW of SFH determined by the ESI-MS was 22999.80 0.48 Da (Fig.3). The obtained MW is in good agreement with the theoretical MW, and the measurement deviation was 0.02 Da (0.000087%) less compared to the theoretical MW.

Fig. 3 3.3 Fibrinolytic activity assay of SFH

As the extension of the N-terminus of the HV leads to complete loss of its anticoagulant activity (Shi et al., 2007; Zhang et al., 2008), the fibrinolytic activity assay which was primarily used to determine the bioactivity of SAK, has become an easy and relatively reliable method to determine the bioactivity of SFH in vitro. As shown in Fig.4, the specific fibrinolytic activities of SFH were approximately 44004.8 IU/mg. This is slightly higher compared to the fibrinolytic activity of SFH in our previous report (Shi et al., 2007), suggesting that the fibrinolytic activity of SFH was not compromised by the present production and purification protocol.

Fig. 4 4 Discussion

In this study, SFH was prepared from recombinant E. coli in a 40-L bioreactor. High-cell-density cultivation of the engineered E. coli BL21 (DE3)/pBV-SFH strain was successfully established by controlling the DO concentration and using the step-increase feeding strategy. This approach was simple, efficient, and did not need special equipment. The recovery of proteins from various recombinant strains is a crucial procedure for the preparation of therapeutic or diagnostic proteins. In this study, the hollow fiber column with the 100 kDa cutoff was first used, which rapidly removed the cell debris and the major contaminants, and greatly facilitated the down-stream purification process. The combination of an expanded bed mode and anion exchange chromatographic step was a very efficient protocol for the purification of the SFH. The overall recovery of the soluble SFH from the E. coli cell lysate was estimated to be greater than 54%, and the volumetric productivity of the pure SFH was about 0.6 g/L. This is the highest productivity ever reported for the production of the staphylokinase-based fusion protein up to now. Furthermore, the process significantly reduced the endotoxin level to below-toxic concentrations. In general, there are several advantages with these approaches. First, SFH without any tag might have no potential negative influence on the bioactivity, and the highly efficient purification protocol could also overcome the shortcomings of affinity chromatography often used for the tag-affinity separation in the manufacture of therapeutic proteins (Labrou, 2003). Second, the addition of isopropyl--D-thiogalactopyranoside (IPTG) or other inducers can be avoided by using temperature-sensitive expression vectors, which not only cut down the production cost, but also avoid the potential contamination caused by the additional inducer. Third, fortunately, the over-expressed SFH was in a soluble and active form, and could be extracted by repeatedly freezing and thawing of the E. coli bodies. This obviated the redundant procedures of denaturation and renaturation of the inclusion bodies in most cases of E. coli as a host organism, especially for over-expression. Fourth, the strategies for high-cell-density cultivation greatly increased the volumetric productivity of SFH without the input of pure oxygen. After purification with the highly efficient protocol, the SFH yield was increased by more than 10 times compared to the low-cell-density cultivation. Thus the production cost was reduced, the manufacture time was shortened, and sufficient target proteins were obtained for preclinical and clinical studies. In summary, we have developed an efficient procedure to produce multi-gram quantities of highly pure, soluble, and biologically active SFH from the periplasm of E. coli. These results pave the way for the mass production of SFH for preclinical and clinical use. Acknowledgements This research was supported by the High-Tech Research and Development Program of China (No.
5

2007AA02Z158). We thank Professors JIANG Zhonghua, HUANG Xiangrui, WANG Haidong and GU Xiaopeng for their helpful advice and assistant during the pilot-scale production. We also thank Professor WANG Jiaxi for revising the manuscript. Compliance with ethics guidelines Conflict of Interest Genshen ZHONG, Aiping YU, Bingxing SHI, Yang LIU, Chutse WU declare that they have no conflict of interest. Human and animal rights, informed consent This article does not contain any studies with human or animal subjects performed by the any of the authors. References Choi J H, Keum K C, Lee S Y (2006). Production of recombinant proteins by high cell density culture of Escherichia coli. Chem Eng Sci, 61:876--885 Fernndez-Lahore H M, Geilenkirchen S, Boldt K, Nagel A, Kula M R, Thmmes J (2000). The influence of cell adsorbent interactions on protein adsorption in expanded beds. J Chromatogr A, 873: 195--208 Johnson B H, Hecht M H (1994). Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology (N/Y), 12: 1357--1360 Johnston W, Cord-Ruwisch R, Cooney M J (2002). Industrial control of recombinant E. coli fed-batch culture: new perspectives on traditional controlled variables. Bioproc Biosyst Eng, 25:111--120 Lee S Y (1996). High cell-density culture of Escherichia coli. Trends Biotechnol, 14(3):98--105 Lin D Q, Kula M R, Liten A, Thmmes J (2003). Stability of expanded beds during the application of crude feedstock. Biotechnol Bioeng, 81: 21--26 Labrou N E (2003). Design and selection of ligands for affinity chromatography. J Chromatogr B, 790: 67--78 Lee J, Lee S Y, Park S, Middelberg A P (1999). Control of fed-batch fermentations. Biotechnol Adv, 17:29--48 Miller G L (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 31:426-428 Manderson D, Dempster R, Chisti Y (2006). A recombinant vaccine against hydatidosis: production of the antigen in Escherichia coli. J Ind Microbiol Biotechnol, 33:173--182 Riesenberg D, Schulz V, Knorre W A, Pohl H D, Korz D, Sanders E A, Ross A, Deckwer W D (1991). High cell density cultivation of Escherichia coli at controlled specific growth rate. J Biotechnol, 20:17--27 Shi B X, Yu A P, Liu Y Y, Li J C, Jin J D, Dong C N, Wu C T (2007). Locally activity-released bifunctional fusion protein enhances antithrombosis and alleviates bleeding risk. J Thromb Thrombolys, 24: 283--292 Shiloach J, Fass R (2005). Growing E. coli to high cell density--a historical perspective on method development. Biotechnol Adv, 23:345--57 Saksela O (1981). Radial caseinolysis in agarose: a simple method for detection of plasminogen activator in the presence of inhibitory substances and serum. Anal Biochem, 111: 276--282 Shi B X, Li J C, Yu A P, Yuan B, Wu C T (2006). Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris. Process Biochem, 41: 2446--2451 Yano T, Kobayashi T, Shimizu S (1978). Fed-batch culture of methanol-utilizing bacterium with DO-stat. J Ferment Technol, 56:416--420 Zhang C L, Yu A P, Yuan B, Dong C N, Yu H Y, Wang L S, Wu C T (2008). Construction and functional evaluation of hirudin derivatives with low bleeding risk. Thromb Haemost, 99:324--30 Zhong G S, Shi B X, Wang H D, Jiang Z H, Wu C T (2006). High cell density cultivation of recombinant Escherichia coli for production of fusion protein Staphylokinase-Hirudin. China biotechnol, 26(9): 11--15 (in Chinese)

Fig. 1

M
90 80 70 D O , D C W (g/l) 60 50 40 30 20 10 0 0 2 4 6 8 10 Time (h) 12 14 16 18 20 1 0
DO DCW Glucose

7 6 R es idual G lucos e (g/l) 5 4 3 2

94 KDa 60 KDa 45 KDa

27 KDa

18 KDa

Fig. 1

Temperature-sensitive production of SFH in high-cell-density cultivation of the recombinant E. coli BL21(DE3). A:

Time course of the DO, DCW and residual glucose concentration in the cultivation process. B: SDS-PAGE analysis of the total cell protein of the engineered E. coli BL21(DE3) bacteria after induction. M: The protein marker. Lanes 1 to 4: the post-induction time of 1, 2, 3, 4 h, respectively. E. coli bodies harvested from 4 mL cultivation broth at different induction time points (1--4 h) were re-suspended in 1 mL Tris-HCl buffer (pH 8.0) and boiled for 10 min, and then the supernatants were collected after centrifugation and used for the SDS-PAGE analysis. Arrowhead indicates the SFH band.

Fig. 2

absorbance at 280 nm

absorbance at 280 nm

time/min

NaCl gradient (%)

time/min

C
97 KD 66 KD 53 KD 36 KD

24 KD

Fig. 2

Purification of SFH from the E. coli lysate on pilot scale. A: The elution profile of SFH on the Streamline Q. SFH

was eluted with a NaCl gradient from 0 to 1 M. The arrowhead indicates the SFH elution peaks, and the peaks were pooled. B: The elution profile of the SFH on the Source 15Q. SFH was eluted with a NaCl gradient from 0 to 0.6 M. SFH was present, as indicated by the arrowhead. In both the panels, the protein monitored by A280 nm was recorded on the left axis and the elution conditions were recorded on the right axis, with the gradient trace laid over the chromatogram. C: SDS-PAGE analysis of the purified SFH. Aliquots of the elution peaks were analyzed on 15% SDS-PAGE. M: the pre-stained protein marker; lane 1: the E. coli lysate; lane 2: pool from the Streamline Q; lane 3: pool from the Source 15Q. The arrowhead indicates the SFH on the gel.

NaCl gradient (%)

Fig. 3

S F H -1
100
A 18 A 19 1278.78 1211.49 A 17 1353.92 A 20 1150.90 A 16 1438.45 A 15 1534.22

A : 22999.80?.48 A: 22999.80 0.48

A 21 1096.25

relative abundance (%)

A 22 1046.39

A 14 1643.75 A 13 1770.11

A 23 1000.97 1353.63

1438.21

A 12 1917.61 1769.91

1917.31 1213.06 1213.71 1643.42 1280.49 1281.05 1353.43 1438.00 1355.76 1440.39 1769.80 1536.41 1646.10 1772.44

A 11 2091.90

0 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100

m/z

Mass (m/z)

Fig. 3 Positive ion ESI mass spectrum of SFH. A series of multi-charged molecular ions were observed, which were labeled with the protonation state (An) and the number of protons (n) attached to the protein molecule. The BiomultiView program was used to determine the molecular mass based on the peptide map of the multi-charged molecular ions. The MW of SFH determined by the ESI-MS was 22999.80 0.48 Da.

Fig. 4

A SAK

SFH

B
Logarithm of fibrinolytic activities (IU)

4.1 3.6 3.1

1.2

lgA=0.9289*lgC+1.8569 R2 =0.9513

2.6 2.1 1.6 1.1 0.6 0.6 0.9

Logarithm of diameter of clear

lgY=0.1452*lgA sak +0.6236 R =0.9702

2

1.15

zone(mm)

1.1

1.05 1 0.95 2.4

2.9

3.4

3.9

Logarithm of fibrinoly tic activities(IU)

1.2 1.5 Logarithm of SFH content (g)

1.8

2.1

Fig. 4

Fibrinolytic activity assay of SFH using SAK as a standard. A: Fibrinolytic activities of SFH were determined by

the clear-zone method in agar plates. SAK was used as a standard and was serially diluted. SAK 1--6 represent various bioactive concentrations (0, 3.125, 6.25, 12.5, 25, and 50 IU/well, respectively). SFH was also serially diluted. Correspondingly, SFH 1--6 represent various protein concentrations (0, 0.0625, 0.125, 0.25, 0.5, and 1.0 g/well, respectively). B: Specific fibrinolytic activity of SFH [The small inset in B is the logarithm of the diameters of the clear zones plotted over the logarithm of the concentration of the step-diluted SAK, and the equation, as regressed, was: lgY = 0.1452lgAsak + 0.6236, (R2 = 0.9702)]. Based on this equation, the fibrinolytic activities of SFH can be calculated at various protein concentrations according to the diameters of the clear zones, and then the correlation between the SFH protein concentration with the fibrinolytic activities can be deduced and expressed with the following equation: lgA = 0.9289lgC + 1.8568, (R2 = 0.9513).

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