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Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase
Biologicals xxx (xxxx) xxx–xxx
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Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase Sheethal Thomas Mannullya, V.P.B. Rekhab, N. Singhb, C. Shanthia,∗, K.K. Pulicherlac,∗∗ a
School of Bio Sciences and Technology, Vellore Institute of Technology, Tamil Nadu, Vellore, 632014, India School of Biotechnology, Gautam Buddha University, Greater Noida, UP, India c Department of Science and Technology, Ministry of Science and Technology, Govt. of India, Technology Bavan, New Mehrauli Road, New Delhi, 110016, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipid modification LMSAK Half-life Hydrophobic interaction chromatography Thrombolytic activity Staphylokinase
Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.
1. Introduction Staphylokinase (SAK) is a thrombolytic protein widely used for treating acute myocardial infarction, a prominent cardiovascular disease in the western world [1–5]. The susceptibility to myocardial infarction is increasing significantly among people in developing countries due to changes in lifestyle, bringing the need for thrombolytic proteins with better efficiencies. It is only in the last decade SAK has achieved considerable attention even though, the profibrinolytic properties of SAK was known for more than 50 years [6]. SAK with less half-life has unprecedented potential and offers many benefits in terms of its size, specificity and function when compared with its counterparts- streptokinase, urokinase and tissue plasminogen activator. SAK is smaller in size comprising 136 amino acids in a single polypeptide chain without any disulphide bridges [7,8]. The fibrinolysis induced by SAK does not deplete fibrinogen in plasma and is highly specific when compared with other plasminogen activators [9–11]. Relatively shorter initial half-life of about 6.3 min and terminal half-life about 37 min makes it necessary for continuous infusion of SAK [12,13]. An effective strategy focussed on using SAK therapy has to rely upon its prolonged half-life, stability and reduced immunogenicity. Since SAK is one among the thrombolytic proteins having higher
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potential in treating myocardial infarction [8], research has been focussed on enhancing its thrombolytic properties. SAK has been modified by PEGylation, site specific mutation and lipid modification for making it effective in terms of therapy [5,14–17]. Though purification of modified SAK from various recombinant strains has been reported, different feasible purification methods have been adapted depending upon the various strategies applied to enhance the therapeutic properties of protein [18]. Quantitative amounts of purified protein are required to evaluate its therapeutic and diagnostic efficiency. This requires the purification to be less cumbersome while promoting additional yield. Further the purification process needs to be standardized for a drug in clinical usage. Earlier experiments with native as well as lipobox attached SAK gene cloned and expressed in E. coli GJ1158 have been reported [17]. This pharmacokinetic work is focussed on purification of LMSAK and analysing its thrombolytic efficiency, in terms of half-life. Lipid modified SAK expressed in E. coli GJ1158 under T7 promoter was partially purified using ammonium sulphate precipitation followed by a scalable two step purification method involving hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). The purified protein used for developing polyclonal antibody for pharmacokinetic studies confirmed increase in half-life for LMSAK.
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Shanthi), [email protected] (K.K. Pulicherla).
∗∗
https://doi.org/10.1016/j.biologicals.2020.01.009 Received 19 September 2019; Received in revised form 24 January 2020; Accepted 28 January 2020 1045-1056/ © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Sheethal Thomas Mannully, et al., Biologicals, https://doi.org/10.1016/j.biologicals.2020.01.009
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Fig. 1. Gel analysis of supernatants and pellets of SAK and LMSAK precipitated at various ammonium sulphate concentrations. (a) Lane M: protein marker; lane 1 and 3: 15% precipitated pellet of SAK and LMSAK respectively; lane 2 and 4: 15% precipitated supernatant of SAK and LMSAK respectively. (b) Lane 1 and 3: 20% precipitated supernatant SAK; lane 2 and 4: 20% precipitated pellet of SAK. (c) Lane 1 and 3: 20% precipitated supernatant LMSAK; lane 2 and 4: 20% precipitated pellet of LMSAK. The arrow indicates the (NH4)2SO4 precipitated SAK and LMSAK.
Fig. 2. HIC and SEC purification chromatograms of SAK and LMSAK. Bold line indicates milli-Absorbance Units (mAu) at 280 nm. Dotted line indicates the percentage of 50 mM phosphate buffer (% B) used for the elution of SAK and LMSAK. Arrows indicates the position of SAK and LMSAK. (a) 86th fraction represent SAK; (b) 84th fraction represent LMSAK; 90th to 94th fraction represent other proteins; 111th to 115th fraction represent other high molecular weight proteins; (c) 21st fraction represent SAK; (d) 24th fraction represent LMSAK; 31st to 33rd fraction represent other low molecular weight proteins.
2. Materials and methods
2.2. SAK and LMSAK separation using ammonium sulphate precipitation
2.1. Materials
The recombinant SAK and LMSAK are expressed in E. coli GJ1158 as detailed in an earlier study [17]. For the maximum possible extraction of SAK and LMSAK, expressed cell pellets were resuspended with different buffers at different pH (50 mM acetate buffer, pH 5; 50 mM phosphate buffer, pH 6 and 7; 50 mM tris buffer, pH 8, 9 and 10) and later sonicated with 20 s pulse on and off cycles for 20 min at 4 °C. Cell lysate was then centrifuged at 15294 g for 20 min at 4 °C. Supernatant was subjected to 12% SDS-PAGE to visualize the solubilized protein. The amount of precipitation of the proteins in the cell lysate of SAK and
Hitrap butyl FF column from GE Healthcare (USA), Sephadex G-50 and protein A sepharose from Sigma Aldrich (USA) were used. Freund's adjuvant complete and incomplete, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and bovine serum albumin (BSA) were obtained from Sigma Aldrich (India). The media ingredients used for this study were of analytical grade.
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LMSAK were studied in various concentrations (10%, 20% 30%, 40% and 50%) of (NH4)2SO4. The precipitated proteins were collected by centrifugation at 15294g for 20 min. Supernatant and pellet resuspended in PBS, pH 7.4 were analysed on 12% SDS-PAGE. 2.3. Purification of SAK and LMSAK expressed in GJ1158 using hydrophobic interaction and size exclusion chromatography SAK and LMSAK are subjected to purification in order to study the pharmacokinetic properties. Soluble proteins in 15% (NH4)2SO4 precipitated supernatant of SAK and LMSAK were collected and purified by HIC based hitrap butyl prepacked column (ÄKTA purifier, GE Healthcare) to minimize host protein impurities. The collected fractions were analysed by 12% SDS-PAGE and visualized using coomassie brilliant blue (CBB) R-250 staining. The active fractions of HIC purified SAK and LMSAK were pooled and further purified by loading onto 2.5 × 15.5 cm column (Amersham-Pharmacia, Biotech, USA) of sephadex G-50 equilibrated with 50 mM phosphate buffer, pH 7. The migrated proteins delayed due to molecular size were collected, the active fractions were pooled, desalted with 10 kDa cut-off dialysis membrane (Sigma, India) and subjected in 12% SDS-PAGE. The total protein concentration was estimated using Bradford method [19]. 2.4. Characterization of purified SAK and LMSAK 2.4.1. Proteolytic activity analysis Proteolytic activity of purified SAK and LMSAK was analysed in 10% native PAGE as per the method decribed by Michels and Clark [20]. SAK and LMSAK mixed with plasma in ratio 1:1 was loaded onto wells. 1.5% azocasein in 50 mM tris-HCl buffer, pH 8 was used as substrate where the final concentration of azocasein in gel was kept as 0.02 μg/mL. 2.4.2. In vitro thrombolytic activity analysis The in vitro analysis for enhanced stability and thrombolytic activity of both HIC and SEC purified LMSAK were confirmed with heated plasma agar plate method as per the protocol detailed in Mannully et al. [17]. The enzymatic activity of HIC purified SAK and LMSAK in U was determined using streptokinase as standard shown in figs1 (Supplementary data 1). Here, about 50 μL of 2 μg of purified SAK and LMSAK was loaded per wells onto 25 mL of heated agar plate containing 0.125 mL plasma.
Fig. 3. (a) HIC purified fractions of SAK and LMSAK in 12% SDS-PAGE. Lane M: protein marker; lane 1 to 5: 80th to 84th fraction of LMSAK respectively; lane 6 to 10: 82nd to 86th fraction of SAK respectively. (b) SEC purified fractions of SAK and LMSAK in 12% SDS-PAGE. Lane M: protein marker; lane 1 to 5: 19th to 24th fraction of LMSAK respectively; lane 6 to 10: 17th to 21st fraction of SAK respectively. (c) Silver staining of HIC and SEC purified SAK and LMSAK in 12% SDS-PAGE. Lane 1 and 5: crude cell lysate of SAK and LMSAK respectively; lane 2 and 6: flow through of SAK and LMSAK in HIC column respectively; lane 3 and 7: HIC purified 86th fraction of SAK and 84th fraction of LMSAK respectively; lane 4 and 8; SEC purified 21st fraction of SAK and 24th fraction of LMSAK respectively. (d) Proteolytic activity of purifed SAK and LMSAK in 10% native PAGE gel containing azocasein. Lane 1: SAK; lane 2: LMSAK. (e) Heated plasma agar plate assay of HIC and SEC purified SAK and LMSAK.
2.4.3. Intrinsic fluorescence analysis The intrinsic fluorescence spectral analysis was performed to check the stability of SAK and LMSAK in presence and absence of BSA. SAK and LMSAK were used as control in the study. The purified SAK and LMSAK (1 mg/mL) was mixed with BSA (1 mg/mL) in 50 mM PBS, pH 7.4 in the ratio 1:1 and incubated at different time interval (0–24 h) at 37 °C. The emission spectra intrinsic tryptophan was analysed in cary eclipse fluorescence spectrophotometer (Agilent Technologies, USA) at an excitation wavelength of 280 nm at room temperature using 1 cm path length cuvette and recorded from 300 to 400 nm. The slit widths for both excitation and emission were kept as 5 nm. The biological
Table 1 Purification chart of SAK and LMSAK. Purification stage
Crude cell lysate Ammonium sulphate HIC Sephadex G-50
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Activity recovery or Yield (%)
Purification fold
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
538.9 168.4 17.4 6
755.2 242.1 24.7 8
64972.5 44181.2 32414.4 18563.6
119407.5 83936.4 65376.5 35119.8
120.6 262.3 1862.8 3093.9
158.1 346.8 2646.8 4389.9
100 64.9 49.9 28.6
100 70.3 54.8 29.4
1 2.2 15.5 25.7
1 2.2 16.8 27.8
3
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Fig. 4. Intrinsic fluorescence spectral analysis of SAK and LMSAK in presence and absence of BSA. (a) SAK; (b) LMSAK; (c) BSA; (d) SAK-BSA; (e) LMSAK-BSA.
adjuvant in 1:1 ratio. After first immunization on day 7, subsequent booster injections were administrated to the animals subcutaneously on days 14, 21, 28 and 35. Blood samples (20 μL) were collected on days 0, 7, 14, 21, 28 and 35 by tail bleeding technique [22]. Antibody titer was checked by indirect ELISA as per previous protocol by Lin et al. [23] where, each of the microtitre plates were coated with 100 μL of buffer containing 0.1 μg of SAK or LMSAK. The mice were terminally bled by open chest intracardial puncture [24], serum was separated and purified using protein A sepharose column as per the protocol described by GE Healthcare [25]. The absorbance at λmax 450 doubled in immunised mice in comparison to control mice and this dilution of 1: 64000 was selected for further study. SAK and LMSAK plasma concentration was determined from a standard curve.
activity of 50 μL of 0.5 μg of SAK and LMSAK in presence and absence of BSA were analysed in heated plasma agar plate assay. 2.4.4. Production of polyclonal serum against SAK and LMSAK The purified protein was used to raise polyclonal antisera. Swiss Albino mice were used for collection of polyclonal antisera against SAK and LMSAK. About 6–8 weeks old mice were housed by providing conventional laboratory conditions as mentioned by Marianno et al. [21]. Animal studies were conducted at Vellore Institute of Technology complying with the animal care and usage committee guidelines provided by the institute (Ethical clearance issue no: VIT/IAEC/13/Feb13/ 17). The mice were given an initial subcutaneous injection 200 μL of 250 μg of purified SAK and LMSAK emulsified in Freund's complete 4
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voltage of 90 V for 90 min. The membrane kept in phosphate buffered saline tween 20 (PBST) with 5% (w/v) skimmed milk overnight at 4 °C was probed with polyclonal antiserum (diluted 1/300 in PBST) against SAK and LMSAK respectively for 2 h at room temperature. The washed membrane was additionally probed with HRP conjugated anti-mouse secondary antibody (Santa Cruz, CA, USA, sc-2005) for 1 h and blots were visualized in chemiluminescence using luminal (Sigma, India) as substrate.
Table 2 Intrinsic fluorescence spectral analysis of SAK and LMSAK. SAK
LMSAK
SAK-BSA
LMSAK-BSA
Hour (h)
λmax
Imax
λmax
Imax
λmax
Imax
λmax
Imax
0 1 2 3 4 5 24
350.14 350.54 350.98 351.10 352.43 353.06 354.64
28.26 27.27 24.13 23.81 22.87 20.91 20.51
347.09 347.98 348.01 348.39 349.09 349.82 350.58
30.26 30.23 29.93 29.51 26.43 24.21 24.05
344 345 345.92 346.21 347.07 348.59 349.51
83.92 80.24 79.47 78.48 77.14 76.41 65.47
337 338.04 338.14 339 340.09 342 344.69
99.30 95.69 94.27 89.92 87.41 85.86 72.47
2.4.6. Pharmacokinetic studies of SAK and LMSAK Three treatment groups, each having six male Swiss Albino mice (n = 6) with mean body weight of 25 ± 0.2 g were treated at the same time with SAK, LMSAK and untreated control. Animals were given single intravenous tail injection of 200 μL of purified SAK solution at a dose of 0.2 mg/25 g body weight. Serial blood samples of 20 μL were collected by tail bleeding at just before injection and 5, 15, 30, 60, 90 and 120 min post injection in 0.5 mL eppendorf tubes and plasma was separated by spinning at 1400 g for 10 min. Supernatant was collected and the protein concentration of SAK and LMSAK in plasma was estimated using sandwich ELISA described by Collen et al. [27] by performing triplicates. As capture antibody 2 μg of purified anti-SAK or anti-LMSAK polyclonal antisera obtained by immunizing mice with SAK and LMSAK were coated on polystyrene microtiter plates for 24 h at 4 °C followed by blocking with 5% skimmed milk at 37 °C for 2 h. To the wells, about 100 μL of diluted serum samples collected at each interval were added followed by incubation at 37 °C for 1 h. About 100 μL of HRP conjugated anti-mouse secondary antibody (Santa Cruz, CA, USA, sc-2005) diluted at 1: 5000 ratio in 5% skimmed milk, were added to each well followed by incubation at 37 °C for 2 h. TMB substrate was added to detect the bound enzyme. 1X PBS at pH 7.4 without any detergents was used for washing.
λmax indicates maximum emission wavelength and Imax indicates maximum emission intensity.
Fig. 5. (a) Poinceau staining of SAK and LMSAK in 12% SDS-PAGE. (b) Western blot analysis of SAK and LMSAK visualized using chemiluminescence. Table 3 Pharmacokinetic chart of SAK and LMSAK. Parameters
Unit
SAK
LMSAK
P value
Dose Lamda (z or β) t1/2 Tmax Cmax C0 Clastobs/Cmax AUC(0-t) AUC(0-inf)obs AUC(0-t)/(0-inf)
mg/kg 1/min min min μg/ml μg/ml ————— (μg/ml)min (μg/ml)min
8 0.03 ± 0.003 21.6 ± 2.1 5 0.6 ± 0.004 1.1 ± 0.02 0.02 ± 0.002 11.8 ± 0.4 12.3 ± 0.5 1.0 ± 0.01
8 0.02 ± 0.001 43.3 ± 3.4 5 1.0 ± 0.01 1.8 ± 0.01 0.04 ± 0.002 25.5 ± 0.4 28.1 ± 0.3 0.9 ± 0.01
———— 0.002 0.002 ———— 0.0001 0.001 0.009 0.001 0.007 0.001
(μg/ml)min2 min (mg)/(μg/ml) (mg)/(μg/ml)/ min (mg)/(μg/ml) (mL/kg) (mL/kg)
256.5 ± 31.4 20.8 ± 1.8 20.2 ± 1.4 0.7 ± 0.03
1250.4 ± 40.6 44.4 ± 1.4 17.7 ± 1.5 0.3 ± 0.003
0.002 0.005 0.131 0.001
13.5 ± 0.7 21 ± 1.6 13.5 ± 0.7
12.6 ± 0.4 20 ± 1.8 12.6 ± 0.4
0.146 0.389 0.146
2.4.6.1. Statistical analysis. All the experiments were conducted in triplicates. Data were expressed as mean ± SEM or as mean ± SD. The pharmacokinetic parameters were determined using non compartmental method (log linear trapezoidal) using PK solver software by plotting mean plasma concentration against time. The important pharmacokinetic parameters as mentioned in Table 3 were studied. The log transformation of pharmacokinetic parameters of SAK and LMSAK was calculated by expressing the ratio of two variables as its difference [28]. The significant difference in the mean of pharmacokinetic parameters of SAK and LMSAK was analysed using unpaired Student's t-test. Statistical significance with two tailed P < 0.05 at 95% confidence interval was considered.
obs
AUMC(0-inf)obs MRT(0-inf)obs Vzobs Clobs Vssobs Vdcal Vsscal
3. Results 3.1. Purification of SAK and LMSAK The expressed protein SAK and LMSAK was dissolved in 50 mM phosphate buffer, pH 7 and was precipitated with varying concentration of (NH4)2SO4 to expose hydrophobic sites of the protein to aid binding to the HIC resin. There was more complete precipitation of SAK and LMSAK at 30% as compared to 20%, indicated by arrows in lane 4 and 2 respectively of Fig. 1b and c. For better retention on HIC column, there is a need for the introduction of small hydrophobic patches on the protein. As there was good binding with proteins in 15% (NH4)2SO4 precipitated supernatant of SAK and LMSAK, these protein was chosen for loading onto the column (Fig. 1a). Both SAK and LMSAK were retained in the hitrap butyl FF HIC column without any loss in flow through. The retained protein was eluted by decreasing the salt concentration from 1 to 0.1 M in 50 mM sodium phosphate buffer pH 7. The purity of the eluted peak corresponding to SAK and LMSAK was confirmed by SDS-PAGE (Fig. 3a). HIC eluted 86th fraction of SAK and 84th fraction of LMSAK (Fig. 2a and b) possessed maximal activity of SAK and LMSAK and were loaded
Lamda z or β-elimination rate contant; t1/2-time required to reach half of the original concentration; Tmax-time required to achieve maximum concentration; Cmax-maximum concentration; AUC0-t-area under the concentration-time curve from time zero to the last measurable concentration of SAK and LMSAK; AUC0-inf-area under the concentration-time curve from time 0 to the time t extrapolated to infinite time; AUMC0-inf-area under the curve from time 0 to the time t extrapolated to infinite time; MRT-mean residence time; Vz-volume of distribution (elimination); Vss-Volume of distribution (steady state); Cl-clearance; Vd-Volume of distribution; Obs-observed; Cal-calculated.
2.4.5. Specificity of polyclonal antisera Western blot analysis with polyclonal antiserum prepared against SAK and LMSAK were performed as described by Sambrook et al. [26]. SAK and LMSAK separated in 12% SDS-PAGE gel was transferred onto nitrocellulose membrane (Amersham, GE Healthcare) at a constant 5
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Fig. 6. Pharmacokinetics of SAK and LMSAK. (a) Plot of concentration versus time (mean ± SD) of SAK and LMSAK in mice. (b) the straight line extrapolated to concentration at time 0.
cross reactivity of polyclonal antisera developed against SAK towards LMSAK and vice versa.
onto a SEC column for further purification (Fig. 2c and d). The protein concentration of SAK and LMSAK determine using Bradford assay was 6 and 8 mg/mL respectively [19]. The SAK and LMSAK purified by HIC, followed by SEC showed improved purity in CBB R-250 (Fig. 3a and b) as well as silver stained 12% SDS-PAGE (Fig. 3c). The protein recovery yield after final purification of SAK and LMSAK was found to be 28.6% with 25.7 purification fold for SAK and 29.4% with 27.8 purification fold for LMSAK (Table 1).
3.2.5. Pharmacokinetic studies of SAK and LMSAK Pharmacokinetic studies in mice were carried out to determine enhanced stability and thrombolytic activity of SAK and LMSAK in vivo. Protein concentration in plasma was determined using purified polyclonal antisera and half-life (t1/2) was analysed (Table 3). The plot of log concentration versus time of SAK and LMSAK in plasma is shown in Fig. 6. The maximum concentration (Cmax) of SAK and LMSAK was 0.6 ± 0.004 and 1.0 ± 0.01 μg/mL respectively in plasma after 5 min post injection. SAK was not detectable after 90 min of post dosing, whereas LMSAK having concentration 0.04 ± 0.002 μg/mL was detectable until 120 min of post dosing. SAK and LMSAK showed a mean residence time (MRT) of 20.8 ± 1.8 and 44.4 ± 1.4 min respectively. The half-life of the lipid modified SAK was found to be increased by 2 fold thus confirming the significant influence of lipid modification in enhancing the half-life of the protein in the blood. Similarly, LMSAK had a 2.29 fold slower plasma clearance (Cl) of 0.3 ± 0.003 (mg)/(μg/ mL)/min in comparison to 0.7 ± 0.03 (mg)/(μg/mL)/min of SAK. Since t1/2 and Cl are interrelated, slow clearance indicates better halflife with more amount of time LMSAK present in plasma. Since the volume of distribution (Vd) and elimination rate constant (β) are invariant which doesn't vary with time and dose, there was no significant difference observed in the mean of volume of distribution (elimination) (Vz), Vd and volume of distribution (steady state) (Vss) of SAK and LMSAK. Since it's a preliminary test, the significant difference in the mean of t1/2, Cl and AUC of SAK and LMSAK which would be responsible for an enhancement in therapeutic efficiency of modified protein was analysed. Since both SAK and LMSAK followed a first order kinetics, non compartmental model was applied because sampling times are very few, but it has given the entire features of kinetic behaviour of molecules where the graph concentration (μg/mL) versus time (min) is declining. Elimination rate constant of SAK and LMSAK was found to be 0.03 ± 0.003 and 0.02 ± 0.001 min−1 respectively and AUC0-t was 11.8 ± 0.4 (μg/mL)min for SAK and 25.5 ± 0.4 (μg/mL)min for LMSAK which was responsible for decrease in Vd of LMSAK. Since variability in data increases with increase in measurement size and its effects are multiplicative, data is not directly used to find out correct transformation, instead log transformation is recommended [28]. Ultimately, all the data were converted into log transformation prior to statistical analysis. Since there is no significant difference observed
3.2. Characterization of SAK and LMSAK 3.2.1. Proteolytic and thrombolytic activity The proteolytic activity of purified SAK and LMSAK was confirmed by white zone in blue background in native PAGE gel containing azocasein (Fig. 3d). The thrombolytic activity of the purified sample was confirmed by heated plasma agar plate method (Fig. 3e). The total thrombolytic activity of HIC in succession with SEC purified SAK and LMSAK was found to be decreasing whereas its specific activity was found to be increasing. 3.2.2. Specific activity The specific activity of SEC purified SAK and LMSAK was found to be 3093.93 and 4389.98 U/mg respectively (Table 1). 3.2.3. Intrinsic fluorescence spectral analysis Fluorescence spectral analysis of intrinsic tryptophan residues in SAK and LMSAK excited at 280 nm helps to determine the nature of protein either as native or denatured which depends on their surrounding hydrophobic or hydrophilic environment. The fluorescence spectral analysis of SAK and LMSAK in presence and absence of albumin is depicted in Fig. 4 and in Table 2. Initially the maximum emission wavelength (λmax) of SAK and LMSAK was found to be 350 and 347 nm respectively which on incubation for 24 h at 37 °C gave a red shift in λmax in both cases. When SAK and LMSAK was incubated with albumin, a blue shift in λmax was observed. The blue shift was more in LMSAK. The activity of the enzyme determined by heated plasma agar plate assay was found to remain stable when incubated with albumin. 3.2.4. Western blot analysis of purified SAK and LMSAK A single band corresponding to the molecular weight of 15.5 kDa for SAK and 16.8 kDa LMSAK was observed using chemiluminescence (Fig. 5). There was no non specific binding of anti-mouse secondary antibody towards both the polyclonal antisera. Similarly, there was no 6
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than SAK. Pharmacokinetic studies of LMSAK indicates the potential advantage of lipid modified SAK in increasing half-life on blood circulation in vivo animal models. The lower clearance of LMSAK from blood circulation depends upon its equilibrium with fibrin, plasminogen, proteins and inhibitors in blood stream [39]. Albumin is an important drug transport protein in the blood [40] and has been reported to increase the serum half-life of therapeutic proteins by binding [41,42]. The non covalent association of therapeutic proteins with albumin in blood enhance the half-life by increasing molecular weight higher than renal clearance threshold [43,44]. The site specific conjugation of fatty acid (palmitic acid) to a therapeutic protein enhanced its binding to human serum albumin (HSA) and prolonged its serum half-life [45]. The lipid moiety attached to SAK probably increased its binding to serum albumin and thereby improved its pharmacokinetic properties. To confirm the stability of LMSAK in blood, the intrinsic fluorescence spectral analysis of SAK and LMSAK in presence and absence of albumin was carried out. In the absence of albumin there was a red shift in λmax indicating unfolding of protein due to destabilization and surface localization of tryptophan residues to hydrophilic environment. A blue shift in λmax was observed in presence of albumin confirming protein stability in both cases (SAK and LMSAK). The blue shift was more pronounced in the case of LMSAK in presence of albumin indicating better stability of the protein and also substanciating the in vivo results on improved serum half-life of LMSAK. Hence, lipid modification of staphylokinase can be used as a potential strategy to enhance therapeutic efficiency in terms of half-life, stability and biological activity.
between Vd, it is unlikely that SAK and LMSAK is distributed differently in the tissues. Since same dose of 8 mg/kg was used for both treatments and Vd of LMSAK is slightly lesser than that of SAK, this is not related to different in tissue distribution of those two treatments. Only there is significant difference observed in AUC, MRT and t1/2 which are related to the prolongation of LMSAK to stay in the blood. 3.3. Discussion Thrombolytic agents are administered for treating acute myocardial infarction in patients [5,29]. Globally 7,500,00 patients are estimated to suffer from thrombotic disorders as high cost, side effects and reduced stability limits the usage of such drugs [5]. Several efforts have been done in the past to overcome these hindrances and to enhance the treatment regimen of thrombolytic agents to make it ideal in terms of efficacy, safety and affordability. Among them, SAK an efficient thrombolytic agent in treating acute myocardial infarction offers enormous possibilities. Lipid modification of SAK is one such possibility and was conducted in an earlier study to enhance its stability and activity [17]. With this idea, for a better yield of lipid modified SAK, this study has been focused on its purification and in vivo characterization. Recombinant protein expressed in E. coli was derived either as soluble protein or insoluble aggregates [30]. Proteins aggregate as inclusion bodies required extensive processing for large scale production, purification and recovery of biologically active recombinant proteins [31]. To avoid this protein misfolding and inclusion body formation in the cytoplasm, recombinant proteins could be localized to periplasmic and membrane fraction by the addition of signal peptide at the N terminal. The signal peptide ‘LAGC’ attached at the N terminal of SAK helps to concentrate the protein in particular compartment and allows for a selective passage across the outer membrane through a proteinconducting Sec-translocon channel in the expressing organism [30,32,33]. Both lipid modified SAK and native SAK expressed in GJ1158 was initially purified using HIC chromatography followed by SEC. These butyl sepharose column containing aliphatic resins was used as an initial purification step to suppress protein aggregation and to promote protein refolding with increasing hydrophobicity. Since N terminal cysteine is lipid modified, LMSAK exhibited higher hydrophobicity compared to unmodified SAK. This resulted in different elution pattern with various column matrices where LMSAK was held for longer time in the column. The proteins were purified to homogeneity and were confirmed by SDS-PAGE. The activity of protein was confirmed by native azocasein PAGE and by heated plasma agar plate assay. HIC purified LMSAK had shown higher specific activity than the SAK indicating better separation of LMSAK in HIC column. Further purification was performed using SEC. Activity analysis showed that lipid modification helps in enhancing thrombolytic activity which was confirmed by increased zone of clearance for LMSAK compared to SAK. Since the additional N terminal lipid modified cysteine at position 15 in LMSAK is biologically significant, proper formation of disulfide bridges is required to maintain protein activity. Localization of LMSAK to periplasm and membrane fraction probably provided an oxidative environment that favoured proper disulphide bond formation and correct protein folding which gives higher protein stability and activity [34,35]. The ELISA and the presented PK data are based on quantifying the SAK and LMSAK plasma antigen level. SAK-plasmin complex binding with fibrin is required for its thrombolytic activity [36]. Though PAI-1 and α2-antiplasmin are two important direct plasma fibrinolysis inhibitors [37], it is emphasised that SAK activity in plasma is not inactivated by these plasminogen activator inhibitors because of the presence of fibrin [36,38]. The data in Table 3 showed that the pharmacokinetic properties of LMSAK and SAK is slightly different at examined concentrations which gave higher half-life for LMSAK compared to SAK. The pharmacokinetic results showed that LMSAK has a half-life of 43.3 ± 3.4 min which was found to be being 2 times higher
Declaration of competing interest The authors declare no conflict of interest with this study. Acknowledgements V.P.B. Rekha thanks the financial support by SERB, DST(SB/YS/LS306/2013), Govt. of India. The authors S. T. Mannully and C. Shanthi thank SBST, VIT, Vellore, for providing facilities to perform this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biologicals.2020.01.009. Author contributions The expression work was conceived and designed by K.K. Pulicherla and supervised by V.P.B. Rekha and N. Singh. The purification work was planned and supervised by C. Shanthi. The experiments were performed by S.T. Mannully. References [1] Collen D, Lijnen HR. Staphylokinase, a fibrin specific plasminogen activator with therapeutic potential. Blood 1994;84:680–6. [2] Gray D. Thrombolysis: past, present, and future. Postgrad Med 2006;82:372–5. https://doi.org/10.1136/pgmj.2005.033266. [3] Chen H, Mo W, Su H, Zhang Y, Song H. Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities. BMC Mol Biol 2007;8:88. https://doi.org/10.1186/1471-21998-88. [4] Liu R, Li D, Qiu R, Lin Q, Zhang G, Ma G, Su Z, Hu T. Preparation, characterization and in vitro bioactivity of N-terminally PEGylated staphylokinase dimmers. Process Biochem 2012;47:41–6. https://doi.org/10.1016/j.procbio.2011.10.004. [5] Vanwetswinkel S, Plaisance S, Zhi-yong Z, Vanlinthout I, Brepoels K, Lasters I, Collen D, Jespers L. Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase. Blood 2000;95. 936-2. [6] Ram KS, Peravali JB, Kumar A, Rao KSSS, Pulicherla KK. Staphylokinase: a boon in medical sciences-review. Mintage J Pharm Med Sci 2013;2. 28-4. [7] Schlott B, Gührs K-H, Hartmann M, Röcker A, Collen D. Staphylokinase requires NH2-terminal proteolysis for plasminogen activation. J Biol Chem 1997;272.
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Biologicals journal homepage: www.elsevier.com/locate/biologicals
Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase Sheethal Thomas Mannullya, V.P.B. Rekhab, N. Singhb, C. Shanthia,∗, K.K. Pulicherlac,∗∗ a
School of Bio Sciences and Technology, Vellore Institute of Technology, Tamil Nadu, Vellore, 632014, India School of Biotechnology, Gautam Buddha University, Greater Noida, UP, India c Department of Science and Technology, Ministry of Science and Technology, Govt. of India, Technology Bavan, New Mehrauli Road, New Delhi, 110016, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipid modification LMSAK Half-life Hydrophobic interaction chromatography Thrombolytic activity Staphylokinase
Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.
1. Introduction Staphylokinase (SAK) is a thrombolytic protein widely used for treating acute myocardial infarction, a prominent cardiovascular disease in the western world [1–5]. The susceptibility to myocardial infarction is increasing significantly among people in developing countries due to changes in lifestyle, bringing the need for thrombolytic proteins with better efficiencies. It is only in the last decade SAK has achieved considerable attention even though, the profibrinolytic properties of SAK was known for more than 50 years [6]. SAK with less half-life has unprecedented potential and offers many benefits in terms of its size, specificity and function when compared with its counterparts- streptokinase, urokinase and tissue plasminogen activator. SAK is smaller in size comprising 136 amino acids in a single polypeptide chain without any disulphide bridges [7,8]. The fibrinolysis induced by SAK does not deplete fibrinogen in plasma and is highly specific when compared with other plasminogen activators [9–11]. Relatively shorter initial half-life of about 6.3 min and terminal half-life about 37 min makes it necessary for continuous infusion of SAK [12,13]. An effective strategy focussed on using SAK therapy has to rely upon its prolonged half-life, stability and reduced immunogenicity. Since SAK is one among the thrombolytic proteins having higher
∗
potential in treating myocardial infarction [8], research has been focussed on enhancing its thrombolytic properties. SAK has been modified by PEGylation, site specific mutation and lipid modification for making it effective in terms of therapy [5,14–17]. Though purification of modified SAK from various recombinant strains has been reported, different feasible purification methods have been adapted depending upon the various strategies applied to enhance the therapeutic properties of protein [18]. Quantitative amounts of purified protein are required to evaluate its therapeutic and diagnostic efficiency. This requires the purification to be less cumbersome while promoting additional yield. Further the purification process needs to be standardized for a drug in clinical usage. Earlier experiments with native as well as lipobox attached SAK gene cloned and expressed in E. coli GJ1158 have been reported [17]. This pharmacokinetic work is focussed on purification of LMSAK and analysing its thrombolytic efficiency, in terms of half-life. Lipid modified SAK expressed in E. coli GJ1158 under T7 promoter was partially purified using ammonium sulphate precipitation followed by a scalable two step purification method involving hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). The purified protein used for developing polyclonal antibody for pharmacokinetic studies confirmed increase in half-life for LMSAK.
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Shanthi), [email protected] (K.K. Pulicherla).
∗∗
https://doi.org/10.1016/j.biologicals.2020.01.009 Received 19 September 2019; Received in revised form 24 January 2020; Accepted 28 January 2020 1045-1056/ © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Sheethal Thomas Mannully, et al., Biologicals, https://doi.org/10.1016/j.biologicals.2020.01.009
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Fig. 1. Gel analysis of supernatants and pellets of SAK and LMSAK precipitated at various ammonium sulphate concentrations. (a) Lane M: protein marker; lane 1 and 3: 15% precipitated pellet of SAK and LMSAK respectively; lane 2 and 4: 15% precipitated supernatant of SAK and LMSAK respectively. (b) Lane 1 and 3: 20% precipitated supernatant SAK; lane 2 and 4: 20% precipitated pellet of SAK. (c) Lane 1 and 3: 20% precipitated supernatant LMSAK; lane 2 and 4: 20% precipitated pellet of LMSAK. The arrow indicates the (NH4)2SO4 precipitated SAK and LMSAK.
Fig. 2. HIC and SEC purification chromatograms of SAK and LMSAK. Bold line indicates milli-Absorbance Units (mAu) at 280 nm. Dotted line indicates the percentage of 50 mM phosphate buffer (% B) used for the elution of SAK and LMSAK. Arrows indicates the position of SAK and LMSAK. (a) 86th fraction represent SAK; (b) 84th fraction represent LMSAK; 90th to 94th fraction represent other proteins; 111th to 115th fraction represent other high molecular weight proteins; (c) 21st fraction represent SAK; (d) 24th fraction represent LMSAK; 31st to 33rd fraction represent other low molecular weight proteins.
2. Materials and methods
2.2. SAK and LMSAK separation using ammonium sulphate precipitation
2.1. Materials
The recombinant SAK and LMSAK are expressed in E. coli GJ1158 as detailed in an earlier study [17]. For the maximum possible extraction of SAK and LMSAK, expressed cell pellets were resuspended with different buffers at different pH (50 mM acetate buffer, pH 5; 50 mM phosphate buffer, pH 6 and 7; 50 mM tris buffer, pH 8, 9 and 10) and later sonicated with 20 s pulse on and off cycles for 20 min at 4 °C. Cell lysate was then centrifuged at 15294 g for 20 min at 4 °C. Supernatant was subjected to 12% SDS-PAGE to visualize the solubilized protein. The amount of precipitation of the proteins in the cell lysate of SAK and
Hitrap butyl FF column from GE Healthcare (USA), Sephadex G-50 and protein A sepharose from Sigma Aldrich (USA) were used. Freund's adjuvant complete and incomplete, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and bovine serum albumin (BSA) were obtained from Sigma Aldrich (India). The media ingredients used for this study were of analytical grade.
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LMSAK were studied in various concentrations (10%, 20% 30%, 40% and 50%) of (NH4)2SO4. The precipitated proteins were collected by centrifugation at 15294g for 20 min. Supernatant and pellet resuspended in PBS, pH 7.4 were analysed on 12% SDS-PAGE. 2.3. Purification of SAK and LMSAK expressed in GJ1158 using hydrophobic interaction and size exclusion chromatography SAK and LMSAK are subjected to purification in order to study the pharmacokinetic properties. Soluble proteins in 15% (NH4)2SO4 precipitated supernatant of SAK and LMSAK were collected and purified by HIC based hitrap butyl prepacked column (ÄKTA purifier, GE Healthcare) to minimize host protein impurities. The collected fractions were analysed by 12% SDS-PAGE and visualized using coomassie brilliant blue (CBB) R-250 staining. The active fractions of HIC purified SAK and LMSAK were pooled and further purified by loading onto 2.5 × 15.5 cm column (Amersham-Pharmacia, Biotech, USA) of sephadex G-50 equilibrated with 50 mM phosphate buffer, pH 7. The migrated proteins delayed due to molecular size were collected, the active fractions were pooled, desalted with 10 kDa cut-off dialysis membrane (Sigma, India) and subjected in 12% SDS-PAGE. The total protein concentration was estimated using Bradford method [19]. 2.4. Characterization of purified SAK and LMSAK 2.4.1. Proteolytic activity analysis Proteolytic activity of purified SAK and LMSAK was analysed in 10% native PAGE as per the method decribed by Michels and Clark [20]. SAK and LMSAK mixed with plasma in ratio 1:1 was loaded onto wells. 1.5% azocasein in 50 mM tris-HCl buffer, pH 8 was used as substrate where the final concentration of azocasein in gel was kept as 0.02 μg/mL. 2.4.2. In vitro thrombolytic activity analysis The in vitro analysis for enhanced stability and thrombolytic activity of both HIC and SEC purified LMSAK were confirmed with heated plasma agar plate method as per the protocol detailed in Mannully et al. [17]. The enzymatic activity of HIC purified SAK and LMSAK in U was determined using streptokinase as standard shown in figs1 (Supplementary data 1). Here, about 50 μL of 2 μg of purified SAK and LMSAK was loaded per wells onto 25 mL of heated agar plate containing 0.125 mL plasma.
Fig. 3. (a) HIC purified fractions of SAK and LMSAK in 12% SDS-PAGE. Lane M: protein marker; lane 1 to 5: 80th to 84th fraction of LMSAK respectively; lane 6 to 10: 82nd to 86th fraction of SAK respectively. (b) SEC purified fractions of SAK and LMSAK in 12% SDS-PAGE. Lane M: protein marker; lane 1 to 5: 19th to 24th fraction of LMSAK respectively; lane 6 to 10: 17th to 21st fraction of SAK respectively. (c) Silver staining of HIC and SEC purified SAK and LMSAK in 12% SDS-PAGE. Lane 1 and 5: crude cell lysate of SAK and LMSAK respectively; lane 2 and 6: flow through of SAK and LMSAK in HIC column respectively; lane 3 and 7: HIC purified 86th fraction of SAK and 84th fraction of LMSAK respectively; lane 4 and 8; SEC purified 21st fraction of SAK and 24th fraction of LMSAK respectively. (d) Proteolytic activity of purifed SAK and LMSAK in 10% native PAGE gel containing azocasein. Lane 1: SAK; lane 2: LMSAK. (e) Heated plasma agar plate assay of HIC and SEC purified SAK and LMSAK.
2.4.3. Intrinsic fluorescence analysis The intrinsic fluorescence spectral analysis was performed to check the stability of SAK and LMSAK in presence and absence of BSA. SAK and LMSAK were used as control in the study. The purified SAK and LMSAK (1 mg/mL) was mixed with BSA (1 mg/mL) in 50 mM PBS, pH 7.4 in the ratio 1:1 and incubated at different time interval (0–24 h) at 37 °C. The emission spectra intrinsic tryptophan was analysed in cary eclipse fluorescence spectrophotometer (Agilent Technologies, USA) at an excitation wavelength of 280 nm at room temperature using 1 cm path length cuvette and recorded from 300 to 400 nm. The slit widths for both excitation and emission were kept as 5 nm. The biological
Table 1 Purification chart of SAK and LMSAK. Purification stage
Crude cell lysate Ammonium sulphate HIC Sephadex G-50
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Activity recovery or Yield (%)
Purification fold
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
SAK
LMSAK
538.9 168.4 17.4 6
755.2 242.1 24.7 8
64972.5 44181.2 32414.4 18563.6
119407.5 83936.4 65376.5 35119.8
120.6 262.3 1862.8 3093.9
158.1 346.8 2646.8 4389.9
100 64.9 49.9 28.6
100 70.3 54.8 29.4
1 2.2 15.5 25.7
1 2.2 16.8 27.8
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Fig. 4. Intrinsic fluorescence spectral analysis of SAK and LMSAK in presence and absence of BSA. (a) SAK; (b) LMSAK; (c) BSA; (d) SAK-BSA; (e) LMSAK-BSA.
adjuvant in 1:1 ratio. After first immunization on day 7, subsequent booster injections were administrated to the animals subcutaneously on days 14, 21, 28 and 35. Blood samples (20 μL) were collected on days 0, 7, 14, 21, 28 and 35 by tail bleeding technique [22]. Antibody titer was checked by indirect ELISA as per previous protocol by Lin et al. [23] where, each of the microtitre plates were coated with 100 μL of buffer containing 0.1 μg of SAK or LMSAK. The mice were terminally bled by open chest intracardial puncture [24], serum was separated and purified using protein A sepharose column as per the protocol described by GE Healthcare [25]. The absorbance at λmax 450 doubled in immunised mice in comparison to control mice and this dilution of 1: 64000 was selected for further study. SAK and LMSAK plasma concentration was determined from a standard curve.
activity of 50 μL of 0.5 μg of SAK and LMSAK in presence and absence of BSA were analysed in heated plasma agar plate assay. 2.4.4. Production of polyclonal serum against SAK and LMSAK The purified protein was used to raise polyclonal antisera. Swiss Albino mice were used for collection of polyclonal antisera against SAK and LMSAK. About 6–8 weeks old mice were housed by providing conventional laboratory conditions as mentioned by Marianno et al. [21]. Animal studies were conducted at Vellore Institute of Technology complying with the animal care and usage committee guidelines provided by the institute (Ethical clearance issue no: VIT/IAEC/13/Feb13/ 17). The mice were given an initial subcutaneous injection 200 μL of 250 μg of purified SAK and LMSAK emulsified in Freund's complete 4
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voltage of 90 V for 90 min. The membrane kept in phosphate buffered saline tween 20 (PBST) with 5% (w/v) skimmed milk overnight at 4 °C was probed with polyclonal antiserum (diluted 1/300 in PBST) against SAK and LMSAK respectively for 2 h at room temperature. The washed membrane was additionally probed with HRP conjugated anti-mouse secondary antibody (Santa Cruz, CA, USA, sc-2005) for 1 h and blots were visualized in chemiluminescence using luminal (Sigma, India) as substrate.
Table 2 Intrinsic fluorescence spectral analysis of SAK and LMSAK. SAK
LMSAK
SAK-BSA
LMSAK-BSA
Hour (h)
λmax
Imax
λmax
Imax
λmax
Imax
λmax
Imax
0 1 2 3 4 5 24
350.14 350.54 350.98 351.10 352.43 353.06 354.64
28.26 27.27 24.13 23.81 22.87 20.91 20.51
347.09 347.98 348.01 348.39 349.09 349.82 350.58
30.26 30.23 29.93 29.51 26.43 24.21 24.05
344 345 345.92 346.21 347.07 348.59 349.51
83.92 80.24 79.47 78.48 77.14 76.41 65.47
337 338.04 338.14 339 340.09 342 344.69
99.30 95.69 94.27 89.92 87.41 85.86 72.47
2.4.6. Pharmacokinetic studies of SAK and LMSAK Three treatment groups, each having six male Swiss Albino mice (n = 6) with mean body weight of 25 ± 0.2 g were treated at the same time with SAK, LMSAK and untreated control. Animals were given single intravenous tail injection of 200 μL of purified SAK solution at a dose of 0.2 mg/25 g body weight. Serial blood samples of 20 μL were collected by tail bleeding at just before injection and 5, 15, 30, 60, 90 and 120 min post injection in 0.5 mL eppendorf tubes and plasma was separated by spinning at 1400 g for 10 min. Supernatant was collected and the protein concentration of SAK and LMSAK in plasma was estimated using sandwich ELISA described by Collen et al. [27] by performing triplicates. As capture antibody 2 μg of purified anti-SAK or anti-LMSAK polyclonal antisera obtained by immunizing mice with SAK and LMSAK were coated on polystyrene microtiter plates for 24 h at 4 °C followed by blocking with 5% skimmed milk at 37 °C for 2 h. To the wells, about 100 μL of diluted serum samples collected at each interval were added followed by incubation at 37 °C for 1 h. About 100 μL of HRP conjugated anti-mouse secondary antibody (Santa Cruz, CA, USA, sc-2005) diluted at 1: 5000 ratio in 5% skimmed milk, were added to each well followed by incubation at 37 °C for 2 h. TMB substrate was added to detect the bound enzyme. 1X PBS at pH 7.4 without any detergents was used for washing.
λmax indicates maximum emission wavelength and Imax indicates maximum emission intensity.
Fig. 5. (a) Poinceau staining of SAK and LMSAK in 12% SDS-PAGE. (b) Western blot analysis of SAK and LMSAK visualized using chemiluminescence. Table 3 Pharmacokinetic chart of SAK and LMSAK. Parameters
Unit
SAK
LMSAK
P value
Dose Lamda (z or β) t1/2 Tmax Cmax C0 Clastobs/Cmax AUC(0-t) AUC(0-inf)obs AUC(0-t)/(0-inf)
mg/kg 1/min min min μg/ml μg/ml ————— (μg/ml)min (μg/ml)min
8 0.03 ± 0.003 21.6 ± 2.1 5 0.6 ± 0.004 1.1 ± 0.02 0.02 ± 0.002 11.8 ± 0.4 12.3 ± 0.5 1.0 ± 0.01
8 0.02 ± 0.001 43.3 ± 3.4 5 1.0 ± 0.01 1.8 ± 0.01 0.04 ± 0.002 25.5 ± 0.4 28.1 ± 0.3 0.9 ± 0.01
———— 0.002 0.002 ———— 0.0001 0.001 0.009 0.001 0.007 0.001
(μg/ml)min2 min (mg)/(μg/ml) (mg)/(μg/ml)/ min (mg)/(μg/ml) (mL/kg) (mL/kg)
256.5 ± 31.4 20.8 ± 1.8 20.2 ± 1.4 0.7 ± 0.03
1250.4 ± 40.6 44.4 ± 1.4 17.7 ± 1.5 0.3 ± 0.003
0.002 0.005 0.131 0.001
13.5 ± 0.7 21 ± 1.6 13.5 ± 0.7
12.6 ± 0.4 20 ± 1.8 12.6 ± 0.4
0.146 0.389 0.146
2.4.6.1. Statistical analysis. All the experiments were conducted in triplicates. Data were expressed as mean ± SEM or as mean ± SD. The pharmacokinetic parameters were determined using non compartmental method (log linear trapezoidal) using PK solver software by plotting mean plasma concentration against time. The important pharmacokinetic parameters as mentioned in Table 3 were studied. The log transformation of pharmacokinetic parameters of SAK and LMSAK was calculated by expressing the ratio of two variables as its difference [28]. The significant difference in the mean of pharmacokinetic parameters of SAK and LMSAK was analysed using unpaired Student's t-test. Statistical significance with two tailed P < 0.05 at 95% confidence interval was considered.
obs
AUMC(0-inf)obs MRT(0-inf)obs Vzobs Clobs Vssobs Vdcal Vsscal
3. Results 3.1. Purification of SAK and LMSAK The expressed protein SAK and LMSAK was dissolved in 50 mM phosphate buffer, pH 7 and was precipitated with varying concentration of (NH4)2SO4 to expose hydrophobic sites of the protein to aid binding to the HIC resin. There was more complete precipitation of SAK and LMSAK at 30% as compared to 20%, indicated by arrows in lane 4 and 2 respectively of Fig. 1b and c. For better retention on HIC column, there is a need for the introduction of small hydrophobic patches on the protein. As there was good binding with proteins in 15% (NH4)2SO4 precipitated supernatant of SAK and LMSAK, these protein was chosen for loading onto the column (Fig. 1a). Both SAK and LMSAK were retained in the hitrap butyl FF HIC column without any loss in flow through. The retained protein was eluted by decreasing the salt concentration from 1 to 0.1 M in 50 mM sodium phosphate buffer pH 7. The purity of the eluted peak corresponding to SAK and LMSAK was confirmed by SDS-PAGE (Fig. 3a). HIC eluted 86th fraction of SAK and 84th fraction of LMSAK (Fig. 2a and b) possessed maximal activity of SAK and LMSAK and were loaded
Lamda z or β-elimination rate contant; t1/2-time required to reach half of the original concentration; Tmax-time required to achieve maximum concentration; Cmax-maximum concentration; AUC0-t-area under the concentration-time curve from time zero to the last measurable concentration of SAK and LMSAK; AUC0-inf-area under the concentration-time curve from time 0 to the time t extrapolated to infinite time; AUMC0-inf-area under the curve from time 0 to the time t extrapolated to infinite time; MRT-mean residence time; Vz-volume of distribution (elimination); Vss-Volume of distribution (steady state); Cl-clearance; Vd-Volume of distribution; Obs-observed; Cal-calculated.
2.4.5. Specificity of polyclonal antisera Western blot analysis with polyclonal antiserum prepared against SAK and LMSAK were performed as described by Sambrook et al. [26]. SAK and LMSAK separated in 12% SDS-PAGE gel was transferred onto nitrocellulose membrane (Amersham, GE Healthcare) at a constant 5
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Fig. 6. Pharmacokinetics of SAK and LMSAK. (a) Plot of concentration versus time (mean ± SD) of SAK and LMSAK in mice. (b) the straight line extrapolated to concentration at time 0.
cross reactivity of polyclonal antisera developed against SAK towards LMSAK and vice versa.
onto a SEC column for further purification (Fig. 2c and d). The protein concentration of SAK and LMSAK determine using Bradford assay was 6 and 8 mg/mL respectively [19]. The SAK and LMSAK purified by HIC, followed by SEC showed improved purity in CBB R-250 (Fig. 3a and b) as well as silver stained 12% SDS-PAGE (Fig. 3c). The protein recovery yield after final purification of SAK and LMSAK was found to be 28.6% with 25.7 purification fold for SAK and 29.4% with 27.8 purification fold for LMSAK (Table 1).
3.2.5. Pharmacokinetic studies of SAK and LMSAK Pharmacokinetic studies in mice were carried out to determine enhanced stability and thrombolytic activity of SAK and LMSAK in vivo. Protein concentration in plasma was determined using purified polyclonal antisera and half-life (t1/2) was analysed (Table 3). The plot of log concentration versus time of SAK and LMSAK in plasma is shown in Fig. 6. The maximum concentration (Cmax) of SAK and LMSAK was 0.6 ± 0.004 and 1.0 ± 0.01 μg/mL respectively in plasma after 5 min post injection. SAK was not detectable after 90 min of post dosing, whereas LMSAK having concentration 0.04 ± 0.002 μg/mL was detectable until 120 min of post dosing. SAK and LMSAK showed a mean residence time (MRT) of 20.8 ± 1.8 and 44.4 ± 1.4 min respectively. The half-life of the lipid modified SAK was found to be increased by 2 fold thus confirming the significant influence of lipid modification in enhancing the half-life of the protein in the blood. Similarly, LMSAK had a 2.29 fold slower plasma clearance (Cl) of 0.3 ± 0.003 (mg)/(μg/ mL)/min in comparison to 0.7 ± 0.03 (mg)/(μg/mL)/min of SAK. Since t1/2 and Cl are interrelated, slow clearance indicates better halflife with more amount of time LMSAK present in plasma. Since the volume of distribution (Vd) and elimination rate constant (β) are invariant which doesn't vary with time and dose, there was no significant difference observed in the mean of volume of distribution (elimination) (Vz), Vd and volume of distribution (steady state) (Vss) of SAK and LMSAK. Since it's a preliminary test, the significant difference in the mean of t1/2, Cl and AUC of SAK and LMSAK which would be responsible for an enhancement in therapeutic efficiency of modified protein was analysed. Since both SAK and LMSAK followed a first order kinetics, non compartmental model was applied because sampling times are very few, but it has given the entire features of kinetic behaviour of molecules where the graph concentration (μg/mL) versus time (min) is declining. Elimination rate constant of SAK and LMSAK was found to be 0.03 ± 0.003 and 0.02 ± 0.001 min−1 respectively and AUC0-t was 11.8 ± 0.4 (μg/mL)min for SAK and 25.5 ± 0.4 (μg/mL)min for LMSAK which was responsible for decrease in Vd of LMSAK. Since variability in data increases with increase in measurement size and its effects are multiplicative, data is not directly used to find out correct transformation, instead log transformation is recommended [28]. Ultimately, all the data were converted into log transformation prior to statistical analysis. Since there is no significant difference observed
3.2. Characterization of SAK and LMSAK 3.2.1. Proteolytic and thrombolytic activity The proteolytic activity of purified SAK and LMSAK was confirmed by white zone in blue background in native PAGE gel containing azocasein (Fig. 3d). The thrombolytic activity of the purified sample was confirmed by heated plasma agar plate method (Fig. 3e). The total thrombolytic activity of HIC in succession with SEC purified SAK and LMSAK was found to be decreasing whereas its specific activity was found to be increasing. 3.2.2. Specific activity The specific activity of SEC purified SAK and LMSAK was found to be 3093.93 and 4389.98 U/mg respectively (Table 1). 3.2.3. Intrinsic fluorescence spectral analysis Fluorescence spectral analysis of intrinsic tryptophan residues in SAK and LMSAK excited at 280 nm helps to determine the nature of protein either as native or denatured which depends on their surrounding hydrophobic or hydrophilic environment. The fluorescence spectral analysis of SAK and LMSAK in presence and absence of albumin is depicted in Fig. 4 and in Table 2. Initially the maximum emission wavelength (λmax) of SAK and LMSAK was found to be 350 and 347 nm respectively which on incubation for 24 h at 37 °C gave a red shift in λmax in both cases. When SAK and LMSAK was incubated with albumin, a blue shift in λmax was observed. The blue shift was more in LMSAK. The activity of the enzyme determined by heated plasma agar plate assay was found to remain stable when incubated with albumin. 3.2.4. Western blot analysis of purified SAK and LMSAK A single band corresponding to the molecular weight of 15.5 kDa for SAK and 16.8 kDa LMSAK was observed using chemiluminescence (Fig. 5). There was no non specific binding of anti-mouse secondary antibody towards both the polyclonal antisera. Similarly, there was no 6
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than SAK. Pharmacokinetic studies of LMSAK indicates the potential advantage of lipid modified SAK in increasing half-life on blood circulation in vivo animal models. The lower clearance of LMSAK from blood circulation depends upon its equilibrium with fibrin, plasminogen, proteins and inhibitors in blood stream [39]. Albumin is an important drug transport protein in the blood [40] and has been reported to increase the serum half-life of therapeutic proteins by binding [41,42]. The non covalent association of therapeutic proteins with albumin in blood enhance the half-life by increasing molecular weight higher than renal clearance threshold [43,44]. The site specific conjugation of fatty acid (palmitic acid) to a therapeutic protein enhanced its binding to human serum albumin (HSA) and prolonged its serum half-life [45]. The lipid moiety attached to SAK probably increased its binding to serum albumin and thereby improved its pharmacokinetic properties. To confirm the stability of LMSAK in blood, the intrinsic fluorescence spectral analysis of SAK and LMSAK in presence and absence of albumin was carried out. In the absence of albumin there was a red shift in λmax indicating unfolding of protein due to destabilization and surface localization of tryptophan residues to hydrophilic environment. A blue shift in λmax was observed in presence of albumin confirming protein stability in both cases (SAK and LMSAK). The blue shift was more pronounced in the case of LMSAK in presence of albumin indicating better stability of the protein and also substanciating the in vivo results on improved serum half-life of LMSAK. Hence, lipid modification of staphylokinase can be used as a potential strategy to enhance therapeutic efficiency in terms of half-life, stability and biological activity.
between Vd, it is unlikely that SAK and LMSAK is distributed differently in the tissues. Since same dose of 8 mg/kg was used for both treatments and Vd of LMSAK is slightly lesser than that of SAK, this is not related to different in tissue distribution of those two treatments. Only there is significant difference observed in AUC, MRT and t1/2 which are related to the prolongation of LMSAK to stay in the blood. 3.3. Discussion Thrombolytic agents are administered for treating acute myocardial infarction in patients [5,29]. Globally 7,500,00 patients are estimated to suffer from thrombotic disorders as high cost, side effects and reduced stability limits the usage of such drugs [5]. Several efforts have been done in the past to overcome these hindrances and to enhance the treatment regimen of thrombolytic agents to make it ideal in terms of efficacy, safety and affordability. Among them, SAK an efficient thrombolytic agent in treating acute myocardial infarction offers enormous possibilities. Lipid modification of SAK is one such possibility and was conducted in an earlier study to enhance its stability and activity [17]. With this idea, for a better yield of lipid modified SAK, this study has been focused on its purification and in vivo characterization. Recombinant protein expressed in E. coli was derived either as soluble protein or insoluble aggregates [30]. Proteins aggregate as inclusion bodies required extensive processing for large scale production, purification and recovery of biologically active recombinant proteins [31]. To avoid this protein misfolding and inclusion body formation in the cytoplasm, recombinant proteins could be localized to periplasmic and membrane fraction by the addition of signal peptide at the N terminal. The signal peptide ‘LAGC’ attached at the N terminal of SAK helps to concentrate the protein in particular compartment and allows for a selective passage across the outer membrane through a proteinconducting Sec-translocon channel in the expressing organism [30,32,33]. Both lipid modified SAK and native SAK expressed in GJ1158 was initially purified using HIC chromatography followed by SEC. These butyl sepharose column containing aliphatic resins was used as an initial purification step to suppress protein aggregation and to promote protein refolding with increasing hydrophobicity. Since N terminal cysteine is lipid modified, LMSAK exhibited higher hydrophobicity compared to unmodified SAK. This resulted in different elution pattern with various column matrices where LMSAK was held for longer time in the column. The proteins were purified to homogeneity and were confirmed by SDS-PAGE. The activity of protein was confirmed by native azocasein PAGE and by heated plasma agar plate assay. HIC purified LMSAK had shown higher specific activity than the SAK indicating better separation of LMSAK in HIC column. Further purification was performed using SEC. Activity analysis showed that lipid modification helps in enhancing thrombolytic activity which was confirmed by increased zone of clearance for LMSAK compared to SAK. Since the additional N terminal lipid modified cysteine at position 15 in LMSAK is biologically significant, proper formation of disulfide bridges is required to maintain protein activity. Localization of LMSAK to periplasm and membrane fraction probably provided an oxidative environment that favoured proper disulphide bond formation and correct protein folding which gives higher protein stability and activity [34,35]. The ELISA and the presented PK data are based on quantifying the SAK and LMSAK plasma antigen level. SAK-plasmin complex binding with fibrin is required for its thrombolytic activity [36]. Though PAI-1 and α2-antiplasmin are two important direct plasma fibrinolysis inhibitors [37], it is emphasised that SAK activity in plasma is not inactivated by these plasminogen activator inhibitors because of the presence of fibrin [36,38]. The data in Table 3 showed that the pharmacokinetic properties of LMSAK and SAK is slightly different at examined concentrations which gave higher half-life for LMSAK compared to SAK. The pharmacokinetic results showed that LMSAK has a half-life of 43.3 ± 3.4 min which was found to be being 2 times higher
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