Design, preparation and activity evaluation of two novel proteins with thrombolysis or/and neuroprotection 
Huan Chen, Danping Zheng, Xiaoyan Liu, Yuanjun Zhu*, Yinye Wang*          
Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China  
 
 
Abstract: We hypothesized that thrombolysis in combination with neuroprotection might have better therapeutic effects for ischemic stroke compared with thrombolysis alone. In order to verify such hypothesis, we designed a protein TBN by fusing NR6 and BH4, which possibly had dual functions of thrombolysis and neuroprotection. NR6 was obtained by introducingtwo RGD motifs to thrombolytic protein AcAP5 to target thrombus. BH4 is the key domain of anti-apoptotic protein Bcl-xL. The DNA fragments encoding TBN and NR6 were synthesized and cloned into pET30a and pET16b vectors, respectively. Both proteins were expressed in E. coli. BL21 (DE3), mainly in the form of inclusion bodies. With His-tag, NR6 was purified by nickel affinity chromatography, while TBN was purified by ion exchange chromatography. Purified proteins were refolded by dialysis assay. The thrombolytic activity of both proteins was evaluated by the rat arteriovenous bypass model. Both NR6 and TBN significantly reduced thrombus weight at higher dose (24 nmol/kg), TBN showed similar effect to NR6. These results suggested that NR6 was a thrombolytic protein, and fusion protein TBN reserved the thrombolytic activation of NR6. The effects of both proteins were also evaluated in thromboembolic middle cerebral artery occlusion (eMCAO) in mice. TBN exhibited better effect on reducing infarction volume and inhibiting apoptosis of cells than NR6, indicating that the introduction of BH4 increased the protective effect of NR6. The hemorrhagic side effects of the two proteins were evaluated by tail bleeding in mice, and it was found that NR6 and TBN showed shorter bleeding time compared with tPA. In conclusion, we designed and prepared the two novel proteins, and testified that they had significant thrombolytic effect and protective effect on cerebral IR injury. The protective effect of TBN was more potent than NR6. Their bleeding side reaction might be weaker than tPA. These results suggested that these two novel proteins deserved to be further investigated as new thrombolytic candidate agents.  
Keywords: Ischemic stroke; Thrombolysis; Neuroprotection; Fusion protein   
CLC number: R962                Document code: A                 Article ID: 10031057(2018)1178712
 
 
1. Introduction
Stroke is a disease with high morbidity, high disability rate and high mortality in the world, and ischemic strokeaccounts for about 80% of the total number of strokes[1].The current clinical treatment of ischemic stroke mainly includes tPA intravenous thrombolysis to restore the blood supply of ischemic area, preventing the further expansion of infarction area[2]. However, the blood restoration can cause reperfusion injury[3]. The mechanisms of cerebral ischemia and reperfusion injury include several aspects as follows: oxidative stress, excitatory amino acid toxicity, blood-brain barrier disruption, inflammatory response, autophagy and apoptosis, etc. Due to ischemia-reperfusion injury, thrombolytic therapy alone may not achieve the desired therapeutic effect.
Apoptosis is an important mechanism in ischemic reperfusion injury, and it is the main form of delayed or secondary death of penumbra neurons[4]. Inhibiting ischemia-induced apoptosis can reduce the ischemic injury of brain. BcL-xL is an anti-apoptosis protein, which prevents the release of cytochrome C from mitochondria and prevents the decline of mitochondrial membrane potential by regulating the activity of the voltage-dependent anion channel. Cao et al. have found that BcL-xL significantly reduces infarct volume in tMCAO mice[5]. BH4 is the functional domain of Bcl-xL, and it exerts similar anti-apoptotic effect with Bcl-xL[6,7]. As BH4 has a much smaller molecular weight than Bcl-xL, BH4 may be used as a neuroprotective unit to react with a thrombolysis agent to reduce cerebral ischemic damage.
tPA treatment in ischemic stroke often results in hemorrhagic transformation by activating MMP-2 and -9. Therefore, novel thrombolytic agents with different mechanisms from tPA may provide unique advantage. Recent study has found that thrombin-activated fibrinolyticinhibitor (TAFIa) in the bodycan degrade the lysine residues from fibrin C-terminal, thus block the formation of tPA, plasminogen and fibrin ternary complex, and consequently inhibit the thrombolytic effect of tPA[8]. Therefore, inhibition of TAFIa can enhance the fibrinolytic activity of tPA in the body, and TAFIa may become the target of new thrombolytic therapy or auxiliary thrombolytic therapy. Several animal models have demonstrated that TAFIa inhibitors can promote fibrinolysis[9,10]. Because TAFI is activated only when thrombus forms, and activated TAFI (TAFIa) plays its role only on the surface of fibrin. Therefore, TAFIa inhibitors only regulate local fibrinolytic activity, and not increase systemic fibrinolytic activity, and bleeding side reaction may be mild.Ancylostoma anticoagulant peptide 5 (AcAP5) is a peptide isolated from A. caninum hookworms, which has potent anticoagulant activity[11]. Previous studies in our laboratory have found that AcAP5 has strong thrombolytic activity, and its thrombolytic activity is associated with the inhibition of TAFIa[12]. Therefore, AcAP5 deserves further development in thrombolysis.
Platelet activation is closely related to the formation of thrombus, GPIIb/IIIa receptor is the main receptor on activated platelet surface, and it mediates platelet aggregation[13]. RGD peptide is a type of short peptide containing arginine-glycine-aspartic acid sequence, which has high affinity to GPIIb/IIIa receptors[14]. With platelettargeting properties, RGD peptide has been widely used for the preparation of targeted thrombolytic drugs[15,16].
In this work, we engineered a novel thrombolytic protein NR6 by introducing two RGD motifs to AcAP5. We also constructed a fusion protein TBN by fusing BH4 with NR6, with TAT sequence at its N-terminal to cross the blood brain barrier. Moreover, we evaluated the thrombolytic effect and cerebral protective effect on ischemia reperfusion of both proteins.
2. Materials and methods
2.1. Animals
Male Sprague-Dawley rats were purchased from Experimental Animal Center of Peking University Health Science Center. Male KM mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental procedures were approved by Institutional Animal Care and Use Committee of Peking University Health Science Center.
2.2. Bacterial strains, plasmids and enzymes
E. coli BL21 (DE3), E. coli DH5α and pEASY-T1 Cloning Kit were purchased from TransGen Biotech Co., Ltd. (China). Plasmid pET-16b and pET-30a were obtained from Novagen Inc. (USA). Pyrobest DNA polymerase, DNA restriction enzymes and T4 DNA ligase were provided by Dalian Takara (China). Thrombin was supplied by Beijing Solarbio Biotech Co., Ltd. (China). rh-tPA was purchased from World Trade East Pharmaceutical Technology Co., Ltd. (China). 
2.3. Construction of expression plasmids
The DNA fragments encoding NR6 and TBN were obtained by PCR, and the 5' and 3'-ends of the DNA fragments were introduced into Nde I and BamH I sites respectively. After amplification, the DNA fragments were purified by agarose gel electrophoresis, and then cloned into pEASY-T1 cloning vector. After confirmedby DNA sequencing, the reconstructed cloning plasmids pEASY-NR6 and pEASY-TBN were digested with Nde I and BamH I. In addition, the DNA fragments were re-cloned into pET-16b and pET-30a, which were digested with Nde I and BamH I, respectively, yielding expression plasmids pET16b-NR6 and pET30a-TBN. Recombinant expression plasmids were then transformed into E. coli BL21 (DE3) competent cells to generate expression strains E. coli BL21 (DE3)/pET16b-NR6 and E. coli BL21 (DE3)/pET30a-TBN.
2.4. Expressions of NR6 and TBN proteins
A single clone of E. coli BL21 (DE3)/pET16b-NR6 or E. coli BL21 (DE3)/pET30a-TBN was grown at 37 °C overnight in 50 mL LB medium containing 100 μg/mL ampicillin or kanamycin, respectively. The cultures were diluted 100-fold with fresh LB medium and grown at 37 °C until OD600 reached 0.6. The cells were induced with 0.005 mM IPTG at 37 °C overnight, and then were harvested by centrifugation at 4000 r/min at room temperature for 20 min. In order to obtain soluble expression, the protein expression was examined at different temperatures (28 and 23 °C). In order to optimize the expressions of NR6 and TBN proteins, the protein expression was examined at different IPTG concentrations (0.005, 0.01, 0.05, 0.1 and 0.5 mM).
2.5. Purification and refolding of NR6 and TBN
The bacterial cells were harvested and re-suspended in PBS. After sonication and centrifugation at 12 000 r/min for 10 min, the cell pellets containing NR6 or TBN protein (inclusion bodies) were washed by the purgationbuffer A (50 mM Tris, 150 mM NaCl, 1% TritonX-100) or purgation buffer B (3 M Urea, 50 mM Tris, 150 mM NaCl, 1% TritonX-100) twice each for 1 h, respectively. Then the NR6 or TBN inclusion bodies were dissolved in denaturing buffer C (8 M Urea, 50 mM Tris, 150 mM NaCl) or denaturing buffer D (8 M Urea, 50 mM citrateacid, 150 mM NaCl) with gentle agitation at room temperature overnight, respectively.
The dissolved NR6 proteins were filtered through a 0.45-μm membrane, and loaded onto a Ni2+-NTA resin column (GE healthcare, USA) with equilibration using buffer (8 M Urea, 50 mM Tris, 150 mM NaCl, 20 mM imidazole). The proteins were then eluted by a stepwise imidazole gradient in elution buffers (8 M Urea, 50 mM Tris, 150 mM NaCl, 50–500 mM imidazole). The eluent containing target proteins was analyzed by 15% SDS-PAGE.
The dissolved TBN proteins were filtered through a 0.45-μm membrane, and loaded onto a cation-exchanged resin column (SP column, GE healthcare, USA) with equilibration using buffer (8 M Urea, 50 mM citrate acid, 150 mM NaCl, pH 3.4). The proteins were then eluted by a stepwise NaCl gradient in elution buffers (8 M Urea, 50 mM citrate acid, 200–1000 mM NaCl, pH 3.4). The eluent containing target proteins was exchanged pH by dialysis until pH reached 8.5 and then loaded onto an anion-exchanged resin column (Q column, GE healthcare, USA) with equilibration using buffer (8 M Urea, 50 mM Tris, 75 mM NaCl, pH 8.5). The proteins were then eluted by a stepwise NaCl gradient in elution buffers (8 M Urea, 50 mM Tris, 100–500 mM NaCl, pH 8.5). The eluent containing target proteins was analyzed by 15% SDS-PAGE.
The purified NR6 and TBN proteins under the denaturingcondition were refolded by dialysis with refolding buffer and deionized water. After renaturation, the target proteins were filtered through a 0.45-μm membrane. The concentration of protein sample was measured by BCA method. The purity of NR6 protein was determined by 15% SDS-PAGE and analyzed by software (Quantity One).
2.6. Cleavage of TBN by thrombin in vitro
After mixing 270 μL of TBN protein with 30 μL of 10× buffer (200 mM Tris, 1.5 M NaCl, pH 8.0), 0.3 μL of thrombin (Solarbio, 1000 U/mL) was added. The TBN protein was digested with thrombin at 32 ºC for 5, 15, 30, 60 and 240 min, respectively. The digested products were detected by 15% SDS-PAGE.
2.7. Evaluation of thrombolytic effects of NR6 and TBN in vivo on rat arteriovenous bypass thrombolysis model
The thrombolytic activity of NR6 and TBN in vivo was examined by a previously described method[17]with slight modifications. Briefly, male SD rats (240–270 g) were anesthetized with 10% chloral hydrate (3.5 mL/kg, i.p.). Both the right carotid artery and left jugular vein were isolated. Blood was collectedfrom the right carotid artery and infused into a cylindrical thrombus-forming glass tube. Subsequently, a stainlesssteel filament helix was put in it immediately. After 15 min, the helix with thrombus was carefully taken out, weighed, and then put into a polyethylene tube. The polyethylene tube was filled with heparin sodium (150 U/mL), which can be inserted between the left jugular vein and the right carotid artery through linking another two polyethylene tubes with a smaller diameter. NS (3 mL/kg) or NR6 (6, 24 nmol/kg) or tPA (400 nmol/kg) was intravenously injected through the polyethylene tube. The group which was given TBN (6, 24 nmol/kg) used rivaroxaban as anticoagulant. The blood flow was circulated through the polyethylene tube for 60 min, then thrombus was taken out, and its final weight was recorded. The reduced thrombus weight, which can represent the thrombolytic potency in vivo, was calculated.
2.8. Evaluation of neuroprotective effects of NR6 and TBN on mouse model of embolic middle cerebral artery occlusion (eMCAO)
The neuroprotective effects of NR6 and TBN were examined by a previously described method[18] with slight modifications. Briefly, the fresh blood of the mouse heart was extracted and drawn into a PE-0402 catheter (siliconized), and then the blood was allowed to stand for 24 h at room temperature (25 ºC) and stored at 4 ºC until use. Male KM mice (2530 g) were anesthetized with 5% chloral hydrate (10 mL/kg, i.p.). The right carotid artery, internal and external carotid arteries were isolated. The thrombus prepared previously was injected into middle cerebral artery through external carotid artery. An hour later, NS (10 mL/kg) or rhM-tPA (700 nmol/kg) or NR6 (37.5, 150 nmol/kg) or TBN (37.5, 150 nmol/kg) was intravenously injected. After 24 h, the mice were anesthetized with chloral hydrate (500 mg/kg, i.p.) and transcardially perfused with NS. Brains were rapidly collected, sectioned into five 2-mm-thick coronal slices, and stained with 1% 2,3,5-triphenyl-tetrazoliumchloride (TTC) at 37 ºC for 30 min. The slices were fixed in 4% formaldehyde overnight and then photographed with a digital camera. The infarction volumes were analyzed using Image J software.
The caspase-3 activity of cortex, hippocampus and striatum was measured following the instruction of commercial kit. Briefly, lysate was added to the tissue from different brain regions and homogenized for 1 min. After centrifugation at 12 000 r/min at 4 ºC for 10 min, the supernatant was collected, and the protein concentration was determined by Bradford method. The caspase-3 in the supernatant can hydrolyze the substrate DEVE-pNA to release pNA, which has maximum absorbance at 405 nm. Therefore the caspase-3 activity was measured by absorbance at 405 nm of reaction system.
2.9. Evaluation of bleeding effect of NR6 and TBN
Male KM mice (3540 g) were anesthetized with 5% chloral hydrate (10 mL/kg, i.p.). NS (10 mL/kg) or rhM-tPA (700 nmol/kg) or NR6 (150 nmol/kg) or TBN (150 nmol/kg) was intravenously injected. After 15 min,a wound around 2 cm away from tail root was cut using a razor blade with blood outflow, and then the blood was dipped by absorbent paper every 5 s until the bleeding ceased.
2.10. Statistical analysis
The data were expressed as means±SEM. Statistical evaluation was performed using the One-way ANOVA, followed by Tukey’s test. P<0.05 was considered to be statistically significant.
3. Results
3.1. Design of the proteins and construction of NR6 and TBN expression plasmids
NR6 protein was engineered by introducing two RGD motifs to AcAP5. TBN was engineered by fusing BH4 to NR6, with TAT sequence at N-terminal and MMP/thrombin cutting site (Fig. 1A). The DNA fragments encoding NR6 and TBN proteins were amplified by PCR (NR6: 255 bp, TBN: 429 bp) (Fig. 1B and 1C). The DNA fragments were then cloned into pEASY-T1 vector and verified by colony PCR and DNA sequencing (data not shown). Subsequently, the DNA fragments were separated from pEASY-NR6 and pEASY-TBN by digestion with Nde I and BamH I (Fig. 1D), and re-cloned into pET-16b and pET-30a vector respectively to generate expression plasmids pET16b-NR6 and pET30a-TBN, which were confirmed by digestion with Nde I and BamH I (Fig. 1E).
 
 

Figure 1. Design of the proteins andconstruction of recombinant plasmids. (A) Design schematic of NR6 and TBN. (B) NR6 DNA fragment amplified by PCR. M: DNA marker. 1: NR6. (C) TBN DNA fragment amplified by PCR. M: DNA marker.1: NR6. 2: TBN. (D) pEASY-NR6 and pEASY-TBN digested by Nde I and BamH I. M: DNA marker. 13: pEASY-NR6. 46: pEASY-TBN. (E) pET16b-NR6 and pET30a-TBN digested by Nde I and BamH I. M: DNA marker.1: pET16b-NR6. 2: pET30a-TBN. 

3.2. Expressions of NR6 and TBN

Expressions of NR6 and TBN proteins were detected by 15% SDS-PAGE. The two recombinant proteins were expressed in the pattern of inclusion bodies when induced by IPTG at 37 °C. In order to obtain soluble expression, different temperatures (28 and 23 °C) were tested, and it showed that there were no soluble expressionwhen induced at 28 and 23 °C (data not shown). Therefore, we determined to express the proteins at 37 °C. In order to optimize the expressions of recombinant proteins, different IPTG concentrations (0.005, 0.01, 0.05, 0.1, 0.5 mM) were tested, and it showed that both proteins could obtain high expression level at the lowest IPTG concentration (0.005 mM, Fig. 2).The yield of recombinant NR6 and TBN proteins was about 40 mg/L and 50 mg/L, respectively.
 
 
Figure 2. Optimization of protein expression. (A) The expression of NR6 induced with different concentrations of IPTG. M: prestained protein marker; 1: whole cell lysate of E. coli BL21 (DE3)/pET-16b; 26: whole cell lysate of induced E. coli BL21 (DE3)/pET16b-NR6 with 0.005, 0.01, 0.05, 0.1, 0.5 mM IPTG, respectively. (B) The expression of TBN induced with different concentrations of IPTG. M: prestained protein marker; 1: whole cell lysate of E. coli BL21 (DE3)/pET-30a; 26: whole cell lysate of induced E. coliBL21 (DE3)/pET30a-TBN with 0.005, 0.01, 0.05, 0.1, 0.5 mM IPTG, respectively. Black arrows indicated NR6 and TBN.  
3.3. Purification and refolding of NR6 and TBN
One-step purification for NR6 was performed using Ni-NTA affinity chromatography. It showed that NR6 protein could be well combined with Ni-NTA resin, and NR6 was not eluted until the imidazole concentration reached 150 mM (Fig. 3A). After purification, the purity of NR6 reached about 85%. Purification for TBN was performed using both cation-exchange and anion-exchange chromatography. When purified using cation-exchange resin, impurity proteins were eluted with 400 mM NaCl buffer, and TBN was eluted with 1 M NaCl buffer. Subsequently, TBN was purified using anion-exchange resin, and purified TBN could be obtained by elution with 400 mM NaCl buffer (Fig. 3B). After purification, the purity of TBN reached about 87%. The purified denaturing proteins were refolded by dialysis. Purified and renatured proteins were collected and stored (Fig. 3C).
 
 
Figure 3. Purification and refolding of the recombinant proteins. (A) Purification of NR6. M: prestained protein marker; 1: inclusion body of NR6; 2: flow through; 34: eluents at 20 mM imidazole buffer; 56: eluents at 50 mM imidazole buffer; 78: eluents at 100 mM imidazole buffer; 910: eluents at 150 mM imidazole buffer. (B) Purification of TBN. M: prestained protein marker; 1: inclusion body of TBN (cation-exchange); 2: flow through (cation-exchange); 3: eluent at 400 mM NaCl buffer (cation-exchange); 4: eluent at 1 M NaCl buffer (cation-exchange); 5: inclusion body of TBN (anion-exchange); 6: flow through (anion-exchange); 7: eluent at 400 mM NaCl buffer (anion-exchange). (C) Renaturation of NR6 and TBN. M: prestained protein marker; 1: renatured and purified NR6; 2: renatured and purified TBN. 
3.4. Cleavage of TBN by thrombin in vitro
To confirm that whether TBN could be cleaved by thrombin into separate BH4 and NR6, we conducted the thrombin digestion experiment in vitro. The digested products were detected by 15% SDS-PAGE. The amount of TBN protein was decreased over time, while the amount of NR6 was increased over time. When TBN was digested with thrombin for 4 h, the TBN could be completely digested (Fig. 4). According to this result in vitro, we speculated that TBN could be cleaved by thrombin when it was injected into body. 
 
 
Figure 4. TBN digestion by thrombin. M: prestained protein marker; 1: purified NR6; 2: purified TBN; 37: digested products at 5, 15, 30, 60, 240 min respectively. 
3.5. Evaluation of thrombolytic effects of NR6 and TBN in vivo on rat arteriovenous bypass thrombolysis model
To evaluate the thrombolytic effect of NR6 and TBN, we conducted rat arteriovenous bypass thrombolysis model. The results showed that NR6 slightly reduced thrombus weight at a low dose (6 nmol/kg), and significantly reduced thrombus weight at higher dose (24 nmol/kg) (Fig. 5A). The thrombolytic effect of TBN was similar to NR6, it slightly reduced thrombus weight at a low dose (6 nmol/kg), and significantly reduced thrombus weight at higher dose (24 nmol/kg) (Fig. 5B). These results indicated that both proteins had thrombolytic effect. Moreover, the function of NR6 was remained intact in fusion protein TBN.
 
 
Figure 5. Thrombolytic effect of NR6 and TBN in arteriovenous bypass thrombolysis model in rats. (A) Thrombolytic effect of NR6 in AV bypass thrombolysis model in rats. (B) Thrombolytic effect of TBN in AV bypass thrombolysis model in rats. *P<0.05 versus vehicle; ***P<0.001 versus vehicle (n = 6 per group). 
3.6. Evaluation of neuroprotective effects of NR6 and TBN on mouse model of embolic middle cerebral artery occlusion (eMCAO)
To evaluate the neuroprotective effects of NR6 and TBN, we conducted mouse embolic middle cerebral artery occlusion model. NR6 did not reduce infarct volume at low dose (37.5 nmol/kg). However, at the same low dose (37.5 nmol/kg), the fusion protein TBN showed significantly decreased infarct volume compared with the vehicle group (Fig. 6B). Both proteins showedprotection of cerebral injury at higher dose (150 nmol/kg). More importantly, mice treated with fusion protein TBN showed much less infarction compared with NR6, indicating a stronger neuroprotective effect through combing thrombolysis and neuroprotection. In addition, 150 nmol/kg TBN showed similar effect with 700 nmol/kg tPA. 
The significantly increased caspase-3 activity in ischemic cerebral cortex indicated that apoptosis contributed to ischemic injury, and treatment with NR6 and TBN partially reversed the significantly increased caspase-3 activity. Moreover, TBN had better effect on decreasing caspase-3 activity than NR6 (Fig. 6C). In ischemic hippocampus, there was a significant increase of caspase-3 activity, which was significantly reversed by both NR6 and TBN treatment (Fig. 6D). In the striatum,there was no significant change of caspase-3 activity among the sham, vehicle, NR6 and TBN groups (Fig. 6E). However, the same tendency was seen in cortex, hippocampus and striatum that NR6 and TBN treatment decreased the caspse-3 activity in eMCAO mice. 
 
 
 
Figure 6. Neuroprotective effects of NR6 and TBN. (A) Representative TTC-stained brain slices 24 h after thromboembolic middle cerebral artery occlusion in mice. The white area represents the infarct section, and the red area represents the surviving tissue. (B) Effect of intravenous injection of NR6 and TBN on infarct volume in eMCAO mice. *P<0.05, ***P<0.001 versus vehicle. ^P<0.05 versus NR6 (150 nmol/kg, n = 12 per group). (C) Effect of intravenous injection of NR6 and TBN on caspase-3 activity of cortex in eMCAO mice. ***P<0.001 versus vehicle. ^P<0.05 versus NR6 (n = 7 per group). (D) Effect of intravenous injection of NR6 and TBN on caspase-3 activity of hippocampus in eMCAO mice. **P<0.01, ***P<0.001 versus vehicle (n = 7 per group). (E) Effect of intravenous injection of NR6 and TBN on caspase-3 activity of striatum in eMCAO mice (n = 7 per group).  
3.7. Evaluation of bleeding effect of NR6 and TBN
To assess the bleeding risk of NR6 and TBN, we performed a mouse tail bleeding experiment. The results showed that tPA could significantly prolong the bleeding time compared with vehicle. NR6 and TBN had similar bleeding time, which was prolonged compared with vehicle group, but significantly shorter than tPA-treated mice. This result indicated that NR6 and TBN might have less bleeding risk than tPA in thrombolysis.
 
 
Figure 7. Effect of NR6 and TBN on tail bleeding time in normal mice. **P<0.01, ***P<0.001 versus vehicle. ^^P<0.01 versus tPA. 
4. Discussion
In this study, NR6 gene was initially cloned into pET-30avector. However, NR6 protein was not expressed in E. coli BL21/pET30a-NR6. Then we substituted pET-30a with pET-16b vector, and obtained high-level expression of NR6. What is the mechanism underlying this phenomenon? The secondary structure of the translation initiation area of mRNA has a strong influence on the translation efficiency, which directly affects foreign proteins expressed in E. coli. The folding free energy of the local secondary structure of mRNA is closely related to the expression amount of exogenous protein, the higher the free energy, the lower the expression amount[19]. Bai C et al. have reduced the free energy of the translation initiation area of mRNA by optimizing its secondary structure, and obtained a high-level expression of protein[20]. The introducing of His-tag coding sequence to NR6 by replacing the vector pET-30a with pET-16b may change the secondary structure of the initial area of the mRNA translation, making the translation efficiency greatly improved.
TBN fusion protein was purified by ion exchange chromatography since it contained no His-tag. In the process of purification using ion exchange, proteins are usually in refolded forms. However, we used a unique method, by which TBN proteins were purified by ion exchange in the forms of inclusion bodies. Urea in a wide range of pH exists in the form of molecules, 8 M urea in pH as low as 2.4, its positive ions concentration is only 160 mM, so the urea does not affect the combination of protein and ion exchange resin[21]. Therefore, TBN was directly purified by ion exchange chromatography with a solution of 8 M urea (Fig. 3B). Recombinant proteins in inclusion bodies can be renatured by many methods, such as dialysis dilution and column chromatography. In this work, NR6 and TBN proteins were refolded by dialysis against refolding buffer and then deionized water. The concentration of urea and the pH of solution gradually changed until it was close to neutral, providing a mild environment for protein refolding.
To date, tPA is one of the few thrombolytic drugs in the treatment of ischemic stroke, and these thrombolytic drugs are all plasminogen activators. Two new thrombolyticproteins associated with TAFIa inhibition were preparedand identified in this study. A number of small molecule TAFIa inhibitors have been reported, among which the imidazole acid TAFIa inhibitor has entered the phase I clinical trial[22]. In most studies, TAFIa inhibitors did not show thrombolytic effect when used alone, and they need to combine with tPA and then enhance the thrombolytic effect of tPA[23,24]. In this study, NR6 and TNB exhibited significant thrombolytic effect in arteriovenous shunt thrombolysis model in rats when they were used alone, respectively, and their effective strengths were both comparable with tPA (Fig. 5). This result suggested that both proteins had potent thrombolytic effect. In embolic MCAO model in mice, the separate application of the two proteins showed thrombolysis effect and neuroprotective effect, and the protective effect of TNB was more potent than NR6 (Fig. 6). These data indicated that thrombolytic protein conjugatedanti-apoptotic fragment provided further neuroprotection through reducing reperfusion injury.
In recent years, with the development of nerve vascular unit concept[25], the combined use of thrombolysis and neuroprotective therapy has received more attention, and some animal experiments have proved that the combined application of thrombolysis drug and neuroprotective agents can obtain better therapeutic effect than either drug alone[26]. Clinical trials have also made certain progress. The joint application of neuroprotective agent and tPA can play a synergistic role[27].
In conclusion, we designed and prepared a novel thrombolytic protein NR6 and a novel fusion protein TBN with thrombolytic and neuroprotective function in the present study. Both of them provided significant thrombolytic effects in AV bypass thrombolysis model in rats. The two novel proteins also exhibited potent thrombolytic and cerebral protective effects on eMCAOmodel in mice, and TNB displayed better effect than NR6. Their bleeding side reaction might be weaker than tPA. These results suggested that the two novel proteins deserved further investigation as new thrombolyticcandidate agents.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 8157333 and 81503060).
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具有溶栓和脑保护作用的融合蛋白的表达、纯化及活性研究
陈欢, 郑丹萍, 刘晓岩, 朱元军*, 王银叶*
北京大学医学部 药学院 分子与细胞药理学系, 北京 100191       
摘要: 为了验证溶栓剂联合神经保护剂治疗可能比单纯溶栓治疗产生更好的治疗效果的假设,通过融合NR6BH4设计了一种具有溶栓和神经保护双重功能的蛋白质TBNNR6是通过将两个RGD序列引入溶栓蛋白AcAP5获得的, BH4是抗凋亡蛋白Bcl-xL的关键结构域。编码TBNNR6DNA合成后分别克隆到pET30apET16b载体中,两种蛋白都在大肠杆菌BL21(DE3)中以包涵体的形式表达。NR6带有His标签,通过镍亲和层析纯化,TBN通过离子交换层析纯化,纯化后的蛋白通过透析使其复性。通过大鼠动静脉旁路模型评价两种蛋白质的溶栓活性,在低剂量(6 nmol/kg), NR6TBN具有一定的降低血栓重量的作用(P<0.05),高剂量(24 nmol/kg)时二者可显著降低血栓重量。并且TBN表现出与NR6相同的溶栓作用,表明NR6在融合蛋白TBN中的功能未受影响。通过小鼠血栓栓塞性大脑中动脉堵塞(eMCAO)模型评价两种蛋白质对脑卒中的治疗效果,结果显示TBN降低梗死体积和抑制细胞凋亡的效果优于NR6。通过小鼠尾出血实验评价目的蛋白的出血副作用,结果发现与tPA相比, NR6TBN的出血时间较短。这些结果证实了我们的假设:溶栓联合神经保护治疗缺血性脑卒中的效果优于单独溶栓治疗。这两个蛋白具有作为新型溶栓候选药进一步开发的潜力。 
关键词: 缺血性脑卒中; 溶栓; 神经保护; 融合蛋白 
 
 
 
Received: 2018-04-27, Revised: 2018-07-23, Accepted: 2018-10-10.
Foundation items: National Natural Science Foundation of China (Grant No. 8157333 and 81503060).
*Corresponding author. Tel.: +86-010-82802652, E-mail: wangyinye@bjmu.edu.cn; zhuyuanjun@bjmu.edu.cn       
 
 
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