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Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris
Protein Expression and Purification 66 (2009) 138–142
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris Daping Yang a, Mingli Peng b, Hua Yang a, Qing Yang a,*, Jianqiang Xu a a b
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China
a r t i c l e
i n f o
Article history: Received 12 December 2008 and in revised form 5 March 2009 Available online 13 March 2009 Keywords: Thrombin-like enzyme Fibrinogenolytic enzyme Thrombosis Expression
a b s t r a c t Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5 mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS–PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen a-chain. The enzyme also degraded fibrinogen b-chain to a lesser extent, while showing no degradation toward the c-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombinlike enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 °C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system. Ó 2009 Elsevier Inc. All rights reserved.
Snake venoms contain a large variety of proteins and peptides [1] that interact with a diversity of proteins involved in the blood coagulation cascade and the fibrinolytic pathway [2,3]. Thrombin-like enzymes from snake venoms are serine proteases exhibiting fibrinogen clotting and fibrinogenlytic activities in vitro. Like thrombin, these proteases could also affect the blood coagulation process. But unlike thrombin, they do not activate factor XIII in vivo, and therefore the fibrin produced from cleavage of fibrinogen by these proteases cannot be cross-linked. Consequently, the resulting fibrin is easily dispersed and more susceptible to degradation by plasmin than the fibrin produced by thrombin [4]. This characteristic feature has led to the development of thrombin-like enzymes as potential clinical agents to treat occlusive thrombi. Several thrombin-like enzymes have been cloned and used in the treatment of deep vein thrombosis and peripheral vascular occlusive diseases [5,6]. Gloshedobin is a highly active thrombinlike enzyme obtained from the venom of the snake Gloydius shedaoensis. It has been applied in the treatment of peripheral arterial circulatory disorder. Although some thrombin-like enzymes purified from snake venoms have been commercially available, the * Corresponding author. Fax: +86 0411 84709687. E-mail address: [email protected] (Q. Yang). 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.03.003
popularized clinical use of thrombin-like enzyme has been limited by the immunologic reaction in patients partially due to trace contaminants in the commercial preparations and the high cost of purification under current bioseparation conditions. This has presented a big economical burden to the patients. The expression of these enzymes in microorganisms is a good alternative to produce more recombinant thrombin-like enzymes that can be easily purified for biochemical study at the molecular level to enable the development of a new drug and biochemical studies. A number of protein therapeutics have been applied in clinical treatment, among which recombinant proteins with fusion partner are commercially available, e.g. AlbuleukinTM [7], AlbutropinTM [8], AlbuferonTM-a [9]. In this regard, the activity accounts the most part. Gloshedobin is coded for by a 780 bp nucleotide DNA. It contains 12 cysteine residues that form six disulfide bonds [10], which make it difficult to express in active form in a bacterial system. The snake venom thrombin-like enzymes had not been expressed at high level in the active form in microbial systems in the literature, even if fusion-partner strategy was used. In Escherichia coli, most of the expressed gloshedobin is present as insoluble protein inside inclusion bodies, and the efficiency of refolding for the protein is low [11]. Soluble form of gloshedobin has been obtained by expressing the enzyme in the yeast Pichia pastoris, but the expres-
D. Yang et al. / Protein Expression and Purification 66 (2009) 138–142
sion level of the protein (10 mg/l) was unsatisfactory [12]. In this study, Hsp70 was used to facilitate the active expression of gloshedobin and to eliminate its degradation by the host’s proteases. Hsp70 is a chaperon that has various important functions, including protein folding and protein activation [13]. It has been expressed in P. pastoris to a high level, and the enzyme exhibits both high activity and stability [14]. Thus Hsp70 was fused to the N-terminus of the gloshedobin to increase the expression of gloshedobin in P. pastoris. The Hsp70-gloshedobin fusion protein was expressed in high level as a functional enzyme. Some preliminary characterizations of the recombinant enzyme were also performed.
Materials and methods
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Transformation of P. pastoris The plasmid pPIC9K/hsp70-TLE was digested with SalI and then the DNA was electroporated into P. pastoris GS115 cells at 1.5 kV, 25 lF and 200 X. After electroporation 1 ml of ice-cold 1 M sorbitol was immediately added to the cells. The cells were spread on MD plates (1.34% YNB, 4 105% biotin, 2% dextrose and 2% agar) and incubated for 3 days at 30 °C. GS115 cells were selected for His+ transformants according to published methods (Invitrogen). The Mut+ and Muts phenotypes of the transformants were evaluated by spotting them onto minimal medium (MM) agar plates (1.34% YNB, 4 105% D-biotin, 1.5% agar) containing either 1% dextrose or 0.5% methanol as the primary carbon source. Selection of secreting clones
Materials P. pastoris strain GS115 and plasmid pPIC9K were obtained from Invitrogen (Beijing, China). T4 DNA ligase, Taq DNA polymerase and all the restriction enzymes were obtained from TaKaRa (Dalian, China). Biotin, thrombin, bovine fibrinogen, Na-p-tosyl-GlyPro-Arg-p-nitroanilide were purchased from sigma. Q-Sepharose Fast Flow, HiPrep 26/10 desalting column and Superdex 200 HR were purchased from Pharmacia (Uppsala, Sweden). Yeast nitrogen base was obtained from DIFCO (USA). 3C protease was from Seebio (Shanghai, China). TM
TM
Genomic integration of Hsp70-gloshedobin of clones from MM plate at the P. pastoris AOX1 loci was screened by colony PCR, The forward primer was 50 AOX1 (50 -GACTGGTTCCAATTGACAAGC-30 ) and the reverse primer was 30 AOX1 (50 -GCAAATGGCATTCTGACA TCC-30 ). The amplified product was then analyzed by agarose electrophoresis and sequenced. Positive colonies were each inoculated into 2 ml of buffered minimal glycerol (BMGY) medium in test tubes and incubated for 24 h followed by addition of methanol (1% at every 12 h intervals) and further incubation for 48 h. After incubation the culture supernatant was tested for aminolytic activity. The clone with highest amidolytic activity was selected.
Construction of recombinant gloshedobin expression vector Amidolytic activity assay The Hsp70 gene was amplified from the vector pPIC9K/hsp70 [14] by polymerase chain reaction (PCR) with the forward primer F1 (50 -GCGGCCGCGCTGGCCTACGGCCTCGACAA-30 ), and the reverse primer R1 (50 -GACGGCCGGGAGGCCAAGGTGGAAGTTCTGTTCCAG GG-30 ). Underline bases correspond to the 3C protease cleavage site in the protein sequence. The gloshedobin DNA was amplified from the vector pET32a(+)/TLE [15] with the forward primer F2 and (50 -AAGTTCTGTTCCAGGGGCCCATCATTGGAGGTGATGAA-30 ) reverse primer R2 (50 -GATGCAACCTGCCCCCA-30 ). The two amplified DNA fragments were then integrated by PCR using forward primer F1 and reverse primer R2. The PCR product containing both hsp70 and gloshedobin gene was purified and digested with XhoI/ EcoRI and then cloned into XhoI/EcoRI cut pPIC9K/hsp70. The resulting construct, named pPIC9K/hsp70-TLE, was used to transform E. coli DH5a. The plasmid was subjected to DNA sequencing to confirm the correct in-frame fusion between the sequence coding for the a-factor secretory signal present in pPIC9K and the sequence of Hsp70-gloshedobin coding region (Fig. 1).
Amidolytic activity was measured by a microplate reader (Tecan, Swiss) using the chromogenic protease substrates Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Assay buffer (200 ll) consisted of 50 mM Tris–HCl buffer (pH 7.5) plus 3 mM chromogenic substrate was incubated for 30 min at room temperature. The reaction was initiated by addition of 20 ll enzyme (1.0 lg), and the release of p-nitroaniline group from the substrate was monitored continuously at 405 nm. One unit of amidolytic activity was defined as the amount of enzyme required to hydrolyze 1.0 lM of substrate per min. Shake flask expression of gloshedobin The selected colone was used to inoculate 100 ml BMGY in a 500 ml flask and incubated at 30 °C with shaking at 250 rpm for 2 days (OD600 of 2–6). The cells were harvested and resuspended in 500 ml BMMY medium in a 2-L flask to an OD600 of 1.0, and then incubated at 28 °C with shaking 250 rpm. A total of six flasks of culture were prepared each having 500 ml. For induction, methanol was added to the culture every 24 h to maintain a concentration of 1% (v/v). Purification of Hsp70-gloshedobin from the P. pastoris supernatants Culture supernatant (3 L) was concentrated to a final volume of 60 ml by ultrafiltration using 10,000 D cellulose ultrafiltration membrane. The concentrated supernatant was subjected to a buffer exchange with 20 mM Tris–HCl (pH 7.5)/20 mM NaCl using HiPrep 26/10 desalting column. The desalted sample was loaded onto XK column (1.6 10 cm) packed with Q-Sepharose Fast Flow and pre-equilibrated with 20 mM Tris–HCl (pH 7.5)/20 mM NaCl (pH 7.5) at a rate of 1.0 ml/min. After washing, the column was eluted with a linear gradient of NaCl from 0 to 0.5 M in 20 mM Tris–HCl (pH 7.5). Fractions were collected at every 4 ml and analyzed for amidolytic activity. The fractions containing major amidolytic activity were pooled and concentrated and further TM
Fig. 1. Map of pPIC9K/hsp70-TLE.
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chromatographed on Superdex 200 HR 10/30 equilibrated with 20 mM Tris–HCl/0.1 M NaCl (pH 7.2). Eluted fractions were assayed for amidolytic activity. Those with major activity were pooled and used as purified enzyme. Protein concentration was determined by BCA method [16] using BSA as standard. TM
amidolytic activity was determined by preincubating 20 ll enzyme (1.0 lg) in 200 ll 50 mM Tris–HCl (pH 7.5) at different temperatures (10, 20, 30, 40, 50, 60, 70 and 80 °C) for 30 min. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). Results and discussion
Electrophoresis and Western blot Construction of expression vector Samples were mixed with an equal volume of 2 loading buffer, heated at 100 °C for 3 min and then electrophorised in 8% SDS-gel. For Western blot analysis protein bands in the gel were blotted onto PVDF membrane. The membrane was blocked with 5% defated milk in TBS for 1 h at room temperature and then incubated with anti-snake venom horse serum (1:500) for 1 h at room temperature. The membrane was washed six times in TBS-T for 10 min each. After washing it was incubated with rabbit anti-horse antibody conjugated with horseradish peroxidase (1:5000) for 30 min at room temperature followed by six washes in TBS-T for 10 min per wash. The blot was developed by the addition of 3,3diaminobenzidine tetrahydrochloride (DAB) at room temperature. Fibrinogenolytic activity assay Fibrinogenolytic activity was determined by incubating 5 ll of enzyme (0.25 lg) with 20 ll fibrinogen (0.4%, w/v) in 20 mM Tris–HCl (pH 7.5) plus 0.15 M NaCl at 37 °C. Aliquots were taken at 10, 30, 40, 50, 60 and 120 min intervals and separated in 10% SDS-gel to examine the cleavage pattern of fibrinogen. Fibrinogen clotting assay Fibrinogen plates were prepared from fibrinogen (0.7%, w/v) dissolved in 20 mM Tris–HCl (pH 7.5) plus 0.5% (w/v) agarose. Two microliter aliquots of purified Hsp70-gloshedobin were spotted onto the plate and incubated at 37 °C for 6 h. To determine the fibrinogen clotting ability of Hsp70-gloshedobin a reference plate was spotted with 2 ll aliquots of thrombin of different concentrations from 0.625 to 100 U/ml, and incubated at the same temperature and for the same amount of time. After incubation the diameter of the clotting zones from each spot were measured. The measurements from the thrombin plate were used to construct a standard curve, and the clotting activity of Hsp70-gloshedobin was determined based on the standard curve and expressed as the equivalent of thrombin units per mg protein.
We have previously expressed recombinant gloshedobin in P. pastoris as a native protein that was secreted into the medium [12]. However, this initial study was not completely satisfactory since the expression level of the recombinant protein about (10 mg/ml) was relatively low in P. pastoris. Hsp70 has been expressed to a high level in P. pastoris yielding as much as 120 mg/l [14]. It was thought that by fusing gloshedobin to the C-terminus of Hsp70 the expression level and stability of gloshedobin could be improved. The 2680 bp hsp70-TLE DNA fragment consisting of Hsp70 gene and the entire coding region of gloshedobin was inserted into XhoI–EcoRI site of pPIC9K/hsp70 vector [14] to yield the construct pPIC9K/hsp70-TLE. Correct insertion and reading frame of Hsp70-gloshedobin were confirmed by DNA sequencing. Small-scale expression studies in shaker flasks The clone with highest amidolytic activity screened from the His+/Mut+ phenotype transformants (containing AOX1 gene that can grow quickly with methanol as the sole carbon source) was used to study the expression of Hsp70-gloshedobin in 500-ml shaking flask. Western blot analysis of the culture supernatant showed that the protein was recognized specifically by horse anti-snake venom antibody (Fig. 2) confirming that the expressed protein was indeed gloshedobin. It has been shown that the expressed recombinant gloshedobin in P. pastoris has amidolytic activity [12]. Purification of recombinant Hsp70-gloshedobin Under the optimal conditions determined above and after 48 h of induction, 3 L of cell free culture medium containing Hsp70-gloshedobin were collected and purified by two chromatography steps. The fusion protein was purified to a single band of about 98 kDa as shown by SDS–PAGE (Fig. 3). The yield of Hsp70-gloshedobin as
Effect of enzyme inhibitor on the amidolytic activity Inhibition of amidolytic activity by inhibitors was determined by incubating 20 ll (1.0 lg) of enzyme in 50 mM Tris–HCl buffer (pH 7.5) containing 1 mM or 5 mM (PMSF), 10 mM ethylene diamine tetraacetic acid (EDTA),1 10 mM TPCK or 10 mM DTT for 30 min at room temperature. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). Effect of pH and temperature on amidolytic activity The effect of pH on amidolytic activity of Hsp70-gloshedobin was determined by assaying the enzyme in the presence of different buffers having different pHs. Assay buffer (200 ll) consisted of 50 mM citrate (pH 3–6) or 50 mM Tris–HCl (pH 7–9) or 50 mM CAPS (pH 10–11) plus 20 ll (1.0 lg) enzyme was preincubated at room temperature for 30 min. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). The effect of temperatures on 1
Abbreviation used: EDTA, ethylene diamine tetraacetic acid.
Fig. 2. SDS–PAGE and Western blot analysis of Hsp70-gloshedobin in P. pastoris harbouring pPIC9K or pPIC9K/hsp70-TLE was induced by methanol to allow expression of Hsp70-gloshedobin. After induction, proteins in the culture supernatant were precipitated with 20% trichloroacetic acid and then subjected to 8% SDSgel followed by Western blot analysis. (A) SDS–PAGE; (B) Western blot. Lane 1, culture supernatant from cells harbouring pPIC9K; lane 2, culture supernatant from cells harbouring pPIC9K/hsp70-TLE.
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Table 2 Inhibition of amidolytic activity of Hsp70-gloshedobin by various protease inhibitors. Inhibitor
Concentration (mM)
Relative activity (%)
Control PMSF PMSF EDTA TLCK DTT Benzamidine
1 5 10 10 10 10
100 38 8.3 98 64.5 54.5 43
purpose to cleave the fusion part was referred mainly to the biochemical studies on gloshedobin itself rather than its function at this moment. Fibrinogen clotting activity
Fig. 3. SDS–PAGE of the different stage of purification of Hsp70-gloshedobin. Lane 1, Fraction from Q-Sepharose FF; lane 2, purified Hsp70-gloshedobin; lane 3, Western blot analysis of the purified enzyme performed with anti-snake venom serum.
The functional activity of Hsp70-gloshedobin was examined by fibrinogen polymerization assay. To perform quantitative analysis of fibrinogen clotting activity of Hsp70-gloshedobin, the clotting activity of Hsp70-gloshedobin was evaluated on the basis of thrombin clotting activity. Hsp70-gloshedobin achieved a fibrinogen clotting activity of 499.8 U/mg, which is lower to that reported for batroxobin (1200 U/mg) [17], but similar to that of ancrod (420 U/mg) [17] and higher than those of jerdonobin (217 U/mg) [18] and crotalase (222 U/mg) [19]. Batroxobin and ancrod are both currently used in the treatment of occlusive thrombi. Fibrinogenlytic activity assay The SDS–PAGE pattern of fibrinogen after digestion with Hsp70gloshedobin is shown in Fig. 4. Under reducing condition, fibrinogen was separated into its subunits of Aa, Bb and c chains. Both the Aa and Bb chains were degraded by Hsp70-gloshedobin, with the Aa chain being more susceptible to degradation, resulting in complete disappearance of the peptide after 1 h of incubation while much of the Bb chain still remained, even with 2 h of incubation. The c chain was resistant to degradation by Hsp70-gloshedobin. From these, we could deduce that Hsp70-gloshedobin induced fibrinogen coagulation through hydrolysis of the Aa and Bb chains. Effect of enzyme inhibitor on amidolytic activity
Fig. 4. Fibrinogenlytic activity of purified Hsp70-gloshedobin. Fibrinogen was incubated with Hsp70-gloshedobin at 37 °C for different time intervals and then subjected to SDS–PAGE. Lane 1, undigested fibrinogen; lanes 2–7, incubation time for fibrinogen plus Hsp70-gloshedobin of 10, 20, 30, 40, 50, 60 and 120 min.
determined by its amidolytic activity was about 67.5% (Table 1), achieving an expression level of 44.5 mg/l which is much higher than that reported for native gloshedobin expressed in P. pastoris [12]. The improvement in protein expression may be due to better protein stability and folding for gloshedobin afforded by Hsp70. However, it is the fact that we failed to remove the fusion partner, Hsp70, from the recombinant gloshedobin by 3C protease, though we did try different digestion conditions. The reason is not uncovered yet and will be investigated in the future work. However, the
In the amidolytic activity analysis, Hsp70-gloshedobin hydrolyzed Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide which is specific to thrombin. Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide is also hydrolyzed by the recombinant gloshedobin expressed without fusion part in P. pastoris [12]. The presence of the fusion part Hsp70 had no affected on the amidolytic activity of the enzyme toward the substrate Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Among various serine proteinase inhibitors tested (Table 2), PMSF was most inhibitory to Hsp70-gloshedobin. DTT, TLCK and Benzamidine showed only partial inhibition. No inhibition was detected with EDTA, a common inhibitor for metalproteinases. The inhibition profile of Hsp70-gloshedobin by protease inhibitors was similar to that of natural thrombin-like enzyme.
Table 1 Purification of recombinant Hsp70-gloshedobin from 3 L of P. pastoris culture supernatant. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Culture supernatant Q-Sepharose FF Superdex 200HR
1467 182.03 90.12
4500 3631.5 3037.5
3.07 19.95 33.70
1 6 11
100 80.7 67.5
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in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. This shows that gloshedobin expressed as a fusion protein with Hsp70 in yeast is a feasible way to obtain a high amount of this enzyme in yeast for different studies, that may eventually lead to the development of a cheaper and yet effective enzyme for treating occlusive thrombi. Acknowledgments We thank Alan K. Chang for critical proof reading of the manuscript. And the financial support provided by the National Key Project for Basic Research (2003CB114400), the National 863 Project (2003AA2Z3520), the Fok Ying Tung Education Foundation, the National Natural Science Foundation of China (20536010) (20576016) (20676021), the State Key Laboratory of Bio-Organic & Natural Products Chemistry (Shanghai), the State Key Laboratory of Bioreactor Engineering (ECUST) (shanghai), the Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian). References
Fig. 5. Effects of different pH and temperatures on amidolytic activity of Hsp70gloshedobin. Amidolytic activity of Hsp70-gloshedobin was conducted in the presence of different pH (A) or temperatures (B) with the substrate Na-p-tosyl-GlyPro-Arg-p-nitroanilide.
The optimum condition for amidolytic activity of Hsp70-gloshedobin The amidolytic activity of Hsp70-gloshedobin showed that the enzyme activity is optimal at a pH of about 7.5 (Fig. 5A) and a temperature of 50 °C (Fig. 5B). About 70% of the enzyme activity was maintained at 70 °C, making it a relatively heat stable enzyme. A distinguishing feature of serine fibrinogenolytic enzymes is that these enzymes can resist inactivation by heat and extreme pH, unlike the fibrinolytic metalloproteinases, which are rapidly inactivated by exposure to extremes of temperature or pH. The heat and pH stabilities of Hsp70-gloshedobin were therefore similar to those of natural thrombin-like enzyme. Conclusion In summary, this is the first report to demonstrate the fusion of Hsp70 with gloshedobin where the fusion has resulted in considerable increase in expression level of the enzyme. The presence of the Hsp70 fusion part had no effect on the properties of gloshedobin including fibrinogen clotting activity, sensitivity to inhibitors, thermal and pH stabilities. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage
[1] T. Matsui, Y. Fujimura, K. Titani, Snake venom proteases affecting hemostasis and thrombosis, Biochim. Biophys. Acta 7 (2000) 146–156. [2] S. Braud, C. Bon, A. Wisner, Snake venom proteins acting on hemostasis, Biochimie 82 (2000) 851–859. [3] H. Pirkel, I. Theodor, Thrombin-like venom enzymes: structure and function, Adv. Exp. Med. Biol. 281 (1990) 165–175. [4] G. Pfeiffer, U. Dabrowski, Carbohydrate structure of a thrombin-like serine protease from Agkistrodin rhodostoma, Eur. J. Biochem. 205 (1992) 961– 978. [5] A.A. Sharp, B.A. Warren, A.M. Paxton, M.J. Allington, Anticoagulant therapy with a purified fraction of Malayan pit viper venom, The Lancet 291 (1968) 493–499. [6] S.K. Tocker, G.H. Barlow, The coagulant enzyme from Bothrops atrox venom (batroxobin), Methods Enzymol. 45 (1976) 214–223. [7] Human genome sciences initiates trial of albuleukinTM, a recombinant human protein for treating solid tumor cancers, HGSI Press, 2002. [8] Results of Phase 1 AlbutropinTM Trial Demonstrate Safety, Prolonged Half-Life and Biological Activity, HGSI Press, 2002. [9] Interim Results of Phase 1 AlbuferonTM-alpha Clinical Trial Demonstrate Safety, Prolonged Half-Life, and Biological Activity in Patients Infected with Hepatitis C, HGSI Press, 2002. [10] Q. Yang, X.Y. Lei, J.Q. Xu, M. Lin, Z.G. Su, L.J. An, J.C. Janson, Cloning and express ion of the cDNA of a thrombin-like enzyme from the venom of Gloydius shedaoensis, J. Dalian Univ. Technol. 43 (2003) 156–159. [11] X. Huang, Q. Yang, M. Yan, Z. Liu, Expression of gloshedobin from the venom of Gloydius shedaoensis in E. coli, Chin. J. Process. Eng. 4 (2004) 22–27. [12] Q. Yang, X.J. Hu, X.M. Xu, X.R. Gao, L.J. An, Z.G. Su, J.C. Janson, Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, Prog. Biochem. Biophys. 29 (2002) 390–393. [13] F.U. Hartl, Molecular chaperones in cellular protein folding, Nature 381 (1996) 571–580. [14] M.L. Peng, N. Ling, H.M. Xu, Y. Qing, R.H. Ling, Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein, Chin. J. Biotechnol. 19 (2003) 286–290. [15] M. Li, Q. Yang, Y.M. Bao, X.Y. Lei, J.Q. Xu, L.J. An, Fusion expression and purification of gloshedobin, a thrombin-like enzyme from Gloydius shedaoensis in E. coli, J. Wuxi Univ. Light Industry 22 (2003) 22–25. [16] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85. [17] H. Pirkle, I. Theodor, D. Miyada, G. Simmons, Thrombin-like enzyme from the venom of Bitis gabonica: purification, properties, and coagulant actions, J. Biol. Chem. 261 (1986) 8830–8835. [18] M.L. Qiu, J. Yang, D.S. Li, W.Y. Wang, Y.L. Xiong, Characterization of a thrombinlike enzyme from the venom of Trimeresurus jerdonii, Toxicon 38 (2000) 1225– 1236. [19] F.S. Markland, P.S. Damus, Purification and properties of a thrombin-like enzyme from the venom of Crotalus adamanteus (Eastern diamondback rattlesnake), J. Biol. Chem. 246 (1971) 6460–6467.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris Daping Yang a, Mingli Peng b, Hua Yang a, Qing Yang a,*, Jianqiang Xu a a b
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China
a r t i c l e
i n f o
Article history: Received 12 December 2008 and in revised form 5 March 2009 Available online 13 March 2009 Keywords: Thrombin-like enzyme Fibrinogenolytic enzyme Thrombosis Expression
a b s t r a c t Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5 mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS–PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen a-chain. The enzyme also degraded fibrinogen b-chain to a lesser extent, while showing no degradation toward the c-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombinlike enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 °C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system. Ó 2009 Elsevier Inc. All rights reserved.
Snake venoms contain a large variety of proteins and peptides [1] that interact with a diversity of proteins involved in the blood coagulation cascade and the fibrinolytic pathway [2,3]. Thrombin-like enzymes from snake venoms are serine proteases exhibiting fibrinogen clotting and fibrinogenlytic activities in vitro. Like thrombin, these proteases could also affect the blood coagulation process. But unlike thrombin, they do not activate factor XIII in vivo, and therefore the fibrin produced from cleavage of fibrinogen by these proteases cannot be cross-linked. Consequently, the resulting fibrin is easily dispersed and more susceptible to degradation by plasmin than the fibrin produced by thrombin [4]. This characteristic feature has led to the development of thrombin-like enzymes as potential clinical agents to treat occlusive thrombi. Several thrombin-like enzymes have been cloned and used in the treatment of deep vein thrombosis and peripheral vascular occlusive diseases [5,6]. Gloshedobin is a highly active thrombinlike enzyme obtained from the venom of the snake Gloydius shedaoensis. It has been applied in the treatment of peripheral arterial circulatory disorder. Although some thrombin-like enzymes purified from snake venoms have been commercially available, the * Corresponding author. Fax: +86 0411 84709687. E-mail address: [email protected] (Q. Yang). 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.03.003
popularized clinical use of thrombin-like enzyme has been limited by the immunologic reaction in patients partially due to trace contaminants in the commercial preparations and the high cost of purification under current bioseparation conditions. This has presented a big economical burden to the patients. The expression of these enzymes in microorganisms is a good alternative to produce more recombinant thrombin-like enzymes that can be easily purified for biochemical study at the molecular level to enable the development of a new drug and biochemical studies. A number of protein therapeutics have been applied in clinical treatment, among which recombinant proteins with fusion partner are commercially available, e.g. AlbuleukinTM [7], AlbutropinTM [8], AlbuferonTM-a [9]. In this regard, the activity accounts the most part. Gloshedobin is coded for by a 780 bp nucleotide DNA. It contains 12 cysteine residues that form six disulfide bonds [10], which make it difficult to express in active form in a bacterial system. The snake venom thrombin-like enzymes had not been expressed at high level in the active form in microbial systems in the literature, even if fusion-partner strategy was used. In Escherichia coli, most of the expressed gloshedobin is present as insoluble protein inside inclusion bodies, and the efficiency of refolding for the protein is low [11]. Soluble form of gloshedobin has been obtained by expressing the enzyme in the yeast Pichia pastoris, but the expres-
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sion level of the protein (10 mg/l) was unsatisfactory [12]. In this study, Hsp70 was used to facilitate the active expression of gloshedobin and to eliminate its degradation by the host’s proteases. Hsp70 is a chaperon that has various important functions, including protein folding and protein activation [13]. It has been expressed in P. pastoris to a high level, and the enzyme exhibits both high activity and stability [14]. Thus Hsp70 was fused to the N-terminus of the gloshedobin to increase the expression of gloshedobin in P. pastoris. The Hsp70-gloshedobin fusion protein was expressed in high level as a functional enzyme. Some preliminary characterizations of the recombinant enzyme were also performed.
Materials and methods
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Transformation of P. pastoris The plasmid pPIC9K/hsp70-TLE was digested with SalI and then the DNA was electroporated into P. pastoris GS115 cells at 1.5 kV, 25 lF and 200 X. After electroporation 1 ml of ice-cold 1 M sorbitol was immediately added to the cells. The cells were spread on MD plates (1.34% YNB, 4 105% biotin, 2% dextrose and 2% agar) and incubated for 3 days at 30 °C. GS115 cells were selected for His+ transformants according to published methods (Invitrogen). The Mut+ and Muts phenotypes of the transformants were evaluated by spotting them onto minimal medium (MM) agar plates (1.34% YNB, 4 105% D-biotin, 1.5% agar) containing either 1% dextrose or 0.5% methanol as the primary carbon source. Selection of secreting clones
Materials P. pastoris strain GS115 and plasmid pPIC9K were obtained from Invitrogen (Beijing, China). T4 DNA ligase, Taq DNA polymerase and all the restriction enzymes were obtained from TaKaRa (Dalian, China). Biotin, thrombin, bovine fibrinogen, Na-p-tosyl-GlyPro-Arg-p-nitroanilide were purchased from sigma. Q-Sepharose Fast Flow, HiPrep 26/10 desalting column and Superdex 200 HR were purchased from Pharmacia (Uppsala, Sweden). Yeast nitrogen base was obtained from DIFCO (USA). 3C protease was from Seebio (Shanghai, China). TM
TM
Genomic integration of Hsp70-gloshedobin of clones from MM plate at the P. pastoris AOX1 loci was screened by colony PCR, The forward primer was 50 AOX1 (50 -GACTGGTTCCAATTGACAAGC-30 ) and the reverse primer was 30 AOX1 (50 -GCAAATGGCATTCTGACA TCC-30 ). The amplified product was then analyzed by agarose electrophoresis and sequenced. Positive colonies were each inoculated into 2 ml of buffered minimal glycerol (BMGY) medium in test tubes and incubated for 24 h followed by addition of methanol (1% at every 12 h intervals) and further incubation for 48 h. After incubation the culture supernatant was tested for aminolytic activity. The clone with highest amidolytic activity was selected.
Construction of recombinant gloshedobin expression vector Amidolytic activity assay The Hsp70 gene was amplified from the vector pPIC9K/hsp70 [14] by polymerase chain reaction (PCR) with the forward primer F1 (50 -GCGGCCGCGCTGGCCTACGGCCTCGACAA-30 ), and the reverse primer R1 (50 -GACGGCCGGGAGGCCAAGGTGGAAGTTCTGTTCCAG GG-30 ). Underline bases correspond to the 3C protease cleavage site in the protein sequence. The gloshedobin DNA was amplified from the vector pET32a(+)/TLE [15] with the forward primer F2 and (50 -AAGTTCTGTTCCAGGGGCCCATCATTGGAGGTGATGAA-30 ) reverse primer R2 (50 -GATGCAACCTGCCCCCA-30 ). The two amplified DNA fragments were then integrated by PCR using forward primer F1 and reverse primer R2. The PCR product containing both hsp70 and gloshedobin gene was purified and digested with XhoI/ EcoRI and then cloned into XhoI/EcoRI cut pPIC9K/hsp70. The resulting construct, named pPIC9K/hsp70-TLE, was used to transform E. coli DH5a. The plasmid was subjected to DNA sequencing to confirm the correct in-frame fusion between the sequence coding for the a-factor secretory signal present in pPIC9K and the sequence of Hsp70-gloshedobin coding region (Fig. 1).
Amidolytic activity was measured by a microplate reader (Tecan, Swiss) using the chromogenic protease substrates Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Assay buffer (200 ll) consisted of 50 mM Tris–HCl buffer (pH 7.5) plus 3 mM chromogenic substrate was incubated for 30 min at room temperature. The reaction was initiated by addition of 20 ll enzyme (1.0 lg), and the release of p-nitroaniline group from the substrate was monitored continuously at 405 nm. One unit of amidolytic activity was defined as the amount of enzyme required to hydrolyze 1.0 lM of substrate per min. Shake flask expression of gloshedobin The selected colone was used to inoculate 100 ml BMGY in a 500 ml flask and incubated at 30 °C with shaking at 250 rpm for 2 days (OD600 of 2–6). The cells were harvested and resuspended in 500 ml BMMY medium in a 2-L flask to an OD600 of 1.0, and then incubated at 28 °C with shaking 250 rpm. A total of six flasks of culture were prepared each having 500 ml. For induction, methanol was added to the culture every 24 h to maintain a concentration of 1% (v/v). Purification of Hsp70-gloshedobin from the P. pastoris supernatants Culture supernatant (3 L) was concentrated to a final volume of 60 ml by ultrafiltration using 10,000 D cellulose ultrafiltration membrane. The concentrated supernatant was subjected to a buffer exchange with 20 mM Tris–HCl (pH 7.5)/20 mM NaCl using HiPrep 26/10 desalting column. The desalted sample was loaded onto XK column (1.6 10 cm) packed with Q-Sepharose Fast Flow and pre-equilibrated with 20 mM Tris–HCl (pH 7.5)/20 mM NaCl (pH 7.5) at a rate of 1.0 ml/min. After washing, the column was eluted with a linear gradient of NaCl from 0 to 0.5 M in 20 mM Tris–HCl (pH 7.5). Fractions were collected at every 4 ml and analyzed for amidolytic activity. The fractions containing major amidolytic activity were pooled and concentrated and further TM
Fig. 1. Map of pPIC9K/hsp70-TLE.
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chromatographed on Superdex 200 HR 10/30 equilibrated with 20 mM Tris–HCl/0.1 M NaCl (pH 7.2). Eluted fractions were assayed for amidolytic activity. Those with major activity were pooled and used as purified enzyme. Protein concentration was determined by BCA method [16] using BSA as standard. TM
amidolytic activity was determined by preincubating 20 ll enzyme (1.0 lg) in 200 ll 50 mM Tris–HCl (pH 7.5) at different temperatures (10, 20, 30, 40, 50, 60, 70 and 80 °C) for 30 min. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). Results and discussion
Electrophoresis and Western blot Construction of expression vector Samples were mixed with an equal volume of 2 loading buffer, heated at 100 °C for 3 min and then electrophorised in 8% SDS-gel. For Western blot analysis protein bands in the gel were blotted onto PVDF membrane. The membrane was blocked with 5% defated milk in TBS for 1 h at room temperature and then incubated with anti-snake venom horse serum (1:500) for 1 h at room temperature. The membrane was washed six times in TBS-T for 10 min each. After washing it was incubated with rabbit anti-horse antibody conjugated with horseradish peroxidase (1:5000) for 30 min at room temperature followed by six washes in TBS-T for 10 min per wash. The blot was developed by the addition of 3,3diaminobenzidine tetrahydrochloride (DAB) at room temperature. Fibrinogenolytic activity assay Fibrinogenolytic activity was determined by incubating 5 ll of enzyme (0.25 lg) with 20 ll fibrinogen (0.4%, w/v) in 20 mM Tris–HCl (pH 7.5) plus 0.15 M NaCl at 37 °C. Aliquots were taken at 10, 30, 40, 50, 60 and 120 min intervals and separated in 10% SDS-gel to examine the cleavage pattern of fibrinogen. Fibrinogen clotting assay Fibrinogen plates were prepared from fibrinogen (0.7%, w/v) dissolved in 20 mM Tris–HCl (pH 7.5) plus 0.5% (w/v) agarose. Two microliter aliquots of purified Hsp70-gloshedobin were spotted onto the plate and incubated at 37 °C for 6 h. To determine the fibrinogen clotting ability of Hsp70-gloshedobin a reference plate was spotted with 2 ll aliquots of thrombin of different concentrations from 0.625 to 100 U/ml, and incubated at the same temperature and for the same amount of time. After incubation the diameter of the clotting zones from each spot were measured. The measurements from the thrombin plate were used to construct a standard curve, and the clotting activity of Hsp70-gloshedobin was determined based on the standard curve and expressed as the equivalent of thrombin units per mg protein.
We have previously expressed recombinant gloshedobin in P. pastoris as a native protein that was secreted into the medium [12]. However, this initial study was not completely satisfactory since the expression level of the recombinant protein about (10 mg/ml) was relatively low in P. pastoris. Hsp70 has been expressed to a high level in P. pastoris yielding as much as 120 mg/l [14]. It was thought that by fusing gloshedobin to the C-terminus of Hsp70 the expression level and stability of gloshedobin could be improved. The 2680 bp hsp70-TLE DNA fragment consisting of Hsp70 gene and the entire coding region of gloshedobin was inserted into XhoI–EcoRI site of pPIC9K/hsp70 vector [14] to yield the construct pPIC9K/hsp70-TLE. Correct insertion and reading frame of Hsp70-gloshedobin were confirmed by DNA sequencing. Small-scale expression studies in shaker flasks The clone with highest amidolytic activity screened from the His+/Mut+ phenotype transformants (containing AOX1 gene that can grow quickly with methanol as the sole carbon source) was used to study the expression of Hsp70-gloshedobin in 500-ml shaking flask. Western blot analysis of the culture supernatant showed that the protein was recognized specifically by horse anti-snake venom antibody (Fig. 2) confirming that the expressed protein was indeed gloshedobin. It has been shown that the expressed recombinant gloshedobin in P. pastoris has amidolytic activity [12]. Purification of recombinant Hsp70-gloshedobin Under the optimal conditions determined above and after 48 h of induction, 3 L of cell free culture medium containing Hsp70-gloshedobin were collected and purified by two chromatography steps. The fusion protein was purified to a single band of about 98 kDa as shown by SDS–PAGE (Fig. 3). The yield of Hsp70-gloshedobin as
Effect of enzyme inhibitor on the amidolytic activity Inhibition of amidolytic activity by inhibitors was determined by incubating 20 ll (1.0 lg) of enzyme in 50 mM Tris–HCl buffer (pH 7.5) containing 1 mM or 5 mM (PMSF), 10 mM ethylene diamine tetraacetic acid (EDTA),1 10 mM TPCK or 10 mM DTT for 30 min at room temperature. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). Effect of pH and temperature on amidolytic activity The effect of pH on amidolytic activity of Hsp70-gloshedobin was determined by assaying the enzyme in the presence of different buffers having different pHs. Assay buffer (200 ll) consisted of 50 mM citrate (pH 3–6) or 50 mM Tris–HCl (pH 7–9) or 50 mM CAPS (pH 10–11) plus 20 ll (1.0 lg) enzyme was preincubated at room temperature for 30 min. The reaction was initiated by addition of 3 mM chromogenic substrate (2 ll). The effect of temperatures on 1
Abbreviation used: EDTA, ethylene diamine tetraacetic acid.
Fig. 2. SDS–PAGE and Western blot analysis of Hsp70-gloshedobin in P. pastoris harbouring pPIC9K or pPIC9K/hsp70-TLE was induced by methanol to allow expression of Hsp70-gloshedobin. After induction, proteins in the culture supernatant were precipitated with 20% trichloroacetic acid and then subjected to 8% SDSgel followed by Western blot analysis. (A) SDS–PAGE; (B) Western blot. Lane 1, culture supernatant from cells harbouring pPIC9K; lane 2, culture supernatant from cells harbouring pPIC9K/hsp70-TLE.
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Table 2 Inhibition of amidolytic activity of Hsp70-gloshedobin by various protease inhibitors. Inhibitor
Concentration (mM)
Relative activity (%)
Control PMSF PMSF EDTA TLCK DTT Benzamidine
1 5 10 10 10 10
100 38 8.3 98 64.5 54.5 43
purpose to cleave the fusion part was referred mainly to the biochemical studies on gloshedobin itself rather than its function at this moment. Fibrinogen clotting activity
Fig. 3. SDS–PAGE of the different stage of purification of Hsp70-gloshedobin. Lane 1, Fraction from Q-Sepharose FF; lane 2, purified Hsp70-gloshedobin; lane 3, Western blot analysis of the purified enzyme performed with anti-snake venom serum.
The functional activity of Hsp70-gloshedobin was examined by fibrinogen polymerization assay. To perform quantitative analysis of fibrinogen clotting activity of Hsp70-gloshedobin, the clotting activity of Hsp70-gloshedobin was evaluated on the basis of thrombin clotting activity. Hsp70-gloshedobin achieved a fibrinogen clotting activity of 499.8 U/mg, which is lower to that reported for batroxobin (1200 U/mg) [17], but similar to that of ancrod (420 U/mg) [17] and higher than those of jerdonobin (217 U/mg) [18] and crotalase (222 U/mg) [19]. Batroxobin and ancrod are both currently used in the treatment of occlusive thrombi. Fibrinogenlytic activity assay The SDS–PAGE pattern of fibrinogen after digestion with Hsp70gloshedobin is shown in Fig. 4. Under reducing condition, fibrinogen was separated into its subunits of Aa, Bb and c chains. Both the Aa and Bb chains were degraded by Hsp70-gloshedobin, with the Aa chain being more susceptible to degradation, resulting in complete disappearance of the peptide after 1 h of incubation while much of the Bb chain still remained, even with 2 h of incubation. The c chain was resistant to degradation by Hsp70-gloshedobin. From these, we could deduce that Hsp70-gloshedobin induced fibrinogen coagulation through hydrolysis of the Aa and Bb chains. Effect of enzyme inhibitor on amidolytic activity
Fig. 4. Fibrinogenlytic activity of purified Hsp70-gloshedobin. Fibrinogen was incubated with Hsp70-gloshedobin at 37 °C for different time intervals and then subjected to SDS–PAGE. Lane 1, undigested fibrinogen; lanes 2–7, incubation time for fibrinogen plus Hsp70-gloshedobin of 10, 20, 30, 40, 50, 60 and 120 min.
determined by its amidolytic activity was about 67.5% (Table 1), achieving an expression level of 44.5 mg/l which is much higher than that reported for native gloshedobin expressed in P. pastoris [12]. The improvement in protein expression may be due to better protein stability and folding for gloshedobin afforded by Hsp70. However, it is the fact that we failed to remove the fusion partner, Hsp70, from the recombinant gloshedobin by 3C protease, though we did try different digestion conditions. The reason is not uncovered yet and will be investigated in the future work. However, the
In the amidolytic activity analysis, Hsp70-gloshedobin hydrolyzed Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide which is specific to thrombin. Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide is also hydrolyzed by the recombinant gloshedobin expressed without fusion part in P. pastoris [12]. The presence of the fusion part Hsp70 had no affected on the amidolytic activity of the enzyme toward the substrate Na-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Among various serine proteinase inhibitors tested (Table 2), PMSF was most inhibitory to Hsp70-gloshedobin. DTT, TLCK and Benzamidine showed only partial inhibition. No inhibition was detected with EDTA, a common inhibitor for metalproteinases. The inhibition profile of Hsp70-gloshedobin by protease inhibitors was similar to that of natural thrombin-like enzyme.
Table 1 Purification of recombinant Hsp70-gloshedobin from 3 L of P. pastoris culture supernatant. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Culture supernatant Q-Sepharose FF Superdex 200HR
1467 182.03 90.12
4500 3631.5 3037.5
3.07 19.95 33.70
1 6 11
100 80.7 67.5
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in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. This shows that gloshedobin expressed as a fusion protein with Hsp70 in yeast is a feasible way to obtain a high amount of this enzyme in yeast for different studies, that may eventually lead to the development of a cheaper and yet effective enzyme for treating occlusive thrombi. Acknowledgments We thank Alan K. Chang for critical proof reading of the manuscript. And the financial support provided by the National Key Project for Basic Research (2003CB114400), the National 863 Project (2003AA2Z3520), the Fok Ying Tung Education Foundation, the National Natural Science Foundation of China (20536010) (20576016) (20676021), the State Key Laboratory of Bio-Organic & Natural Products Chemistry (Shanghai), the State Key Laboratory of Bioreactor Engineering (ECUST) (shanghai), the Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian). References
Fig. 5. Effects of different pH and temperatures on amidolytic activity of Hsp70gloshedobin. Amidolytic activity of Hsp70-gloshedobin was conducted in the presence of different pH (A) or temperatures (B) with the substrate Na-p-tosyl-GlyPro-Arg-p-nitroanilide.
The optimum condition for amidolytic activity of Hsp70-gloshedobin The amidolytic activity of Hsp70-gloshedobin showed that the enzyme activity is optimal at a pH of about 7.5 (Fig. 5A) and a temperature of 50 °C (Fig. 5B). About 70% of the enzyme activity was maintained at 70 °C, making it a relatively heat stable enzyme. A distinguishing feature of serine fibrinogenolytic enzymes is that these enzymes can resist inactivation by heat and extreme pH, unlike the fibrinolytic metalloproteinases, which are rapidly inactivated by exposure to extremes of temperature or pH. The heat and pH stabilities of Hsp70-gloshedobin were therefore similar to those of natural thrombin-like enzyme. Conclusion In summary, this is the first report to demonstrate the fusion of Hsp70 with gloshedobin where the fusion has resulted in considerable increase in expression level of the enzyme. The presence of the Hsp70 fusion part had no effect on the properties of gloshedobin including fibrinogen clotting activity, sensitivity to inhibitors, thermal and pH stabilities. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage
[1] T. Matsui, Y. Fujimura, K. Titani, Snake venom proteases affecting hemostasis and thrombosis, Biochim. Biophys. Acta 7 (2000) 146–156. [2] S. Braud, C. Bon, A. Wisner, Snake venom proteins acting on hemostasis, Biochimie 82 (2000) 851–859. [3] H. Pirkel, I. Theodor, Thrombin-like venom enzymes: structure and function, Adv. Exp. Med. Biol. 281 (1990) 165–175. [4] G. Pfeiffer, U. Dabrowski, Carbohydrate structure of a thrombin-like serine protease from Agkistrodin rhodostoma, Eur. J. Biochem. 205 (1992) 961– 978. [5] A.A. Sharp, B.A. Warren, A.M. Paxton, M.J. Allington, Anticoagulant therapy with a purified fraction of Malayan pit viper venom, The Lancet 291 (1968) 493–499. [6] S.K. Tocker, G.H. Barlow, The coagulant enzyme from Bothrops atrox venom (batroxobin), Methods Enzymol. 45 (1976) 214–223. [7] Human genome sciences initiates trial of albuleukinTM, a recombinant human protein for treating solid tumor cancers, HGSI Press, 2002. [8] Results of Phase 1 AlbutropinTM Trial Demonstrate Safety, Prolonged Half-Life and Biological Activity, HGSI Press, 2002. [9] Interim Results of Phase 1 AlbuferonTM-alpha Clinical Trial Demonstrate Safety, Prolonged Half-Life, and Biological Activity in Patients Infected with Hepatitis C, HGSI Press, 2002. [10] Q. Yang, X.Y. Lei, J.Q. Xu, M. Lin, Z.G. Su, L.J. An, J.C. Janson, Cloning and express ion of the cDNA of a thrombin-like enzyme from the venom of Gloydius shedaoensis, J. Dalian Univ. Technol. 43 (2003) 156–159. [11] X. Huang, Q. Yang, M. Yan, Z. Liu, Expression of gloshedobin from the venom of Gloydius shedaoensis in E. coli, Chin. J. Process. Eng. 4 (2004) 22–27. [12] Q. Yang, X.J. Hu, X.M. Xu, X.R. Gao, L.J. An, Z.G. Su, J.C. Janson, Expression, purification and partial characterization of recombinant gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, Prog. Biochem. Biophys. 29 (2002) 390–393. [13] F.U. Hartl, Molecular chaperones in cellular protein folding, Nature 381 (1996) 571–580. [14] M.L. Peng, N. Ling, H.M. Xu, Y. Qing, R.H. Ling, Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein, Chin. J. Biotechnol. 19 (2003) 286–290. [15] M. Li, Q. Yang, Y.M. Bao, X.Y. Lei, J.Q. Xu, L.J. An, Fusion expression and purification of gloshedobin, a thrombin-like enzyme from Gloydius shedaoensis in E. coli, J. Wuxi Univ. Light Industry 22 (2003) 22–25. [16] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85. [17] H. Pirkle, I. Theodor, D. Miyada, G. Simmons, Thrombin-like enzyme from the venom of Bitis gabonica: purification, properties, and coagulant actions, J. Biol. Chem. 261 (1986) 8830–8835. [18] M.L. Qiu, J. Yang, D.S. Li, W.Y. Wang, Y.L. Xiong, Characterization of a thrombinlike enzyme from the venom of Trimeresurus jerdonii, Toxicon 38 (2000) 1225– 1236. [19] F.S. Markland, P.S. Damus, Purification and properties of a thrombin-like enzyme from the venom of Crotalus adamanteus (Eastern diamondback rattlesnake), J. Biol. Chem. 246 (1971) 6460–6467.