The Effect of Signal Sequence and His-tags on the Expression and Anti-Thrombin Activity of Recombinant Hirudin
-
Shahin Hadadian
,
Leila Pishkar
,
Elmira kazemi
,
Saeme Asgari
,
Mina Sepahi
,
Samira Komijani
,
Parviz Afrough
,
Nazanin Mohajerani
Abstract
Background: Adding signal sequence and His- tags to the expression vectors of recombinant proteins usually increased the production yield by promoting protein secretion to the periplasmic space and by facilitating the purification processes.
In this study, three different expression constructs including vectors with or without signal sequence and His-tags were designed to compare the effect of these elements on Hirudin expression and function. Hirudin is a natural anticoagulant protein produced in the salivary glands of leeches.
Methods: Hirudin variants including cytoplasmic Hirudinn without His-tag (cr-Hirudin), cytoplasmic Hirudin with His-tag (crhis-Hirudin), and periplasmic Hirudin with His-tag (prhis-Hirudin) constructs were expressed in E.coli BL 21(DE3). Ion exchange chromatography and ion metal affinity chromatography were applied for protein purification. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays were performed to assess the anti-thrombin bioactivity of variants.
Results: The expression rate of prhis-Hirudin was approximately 1.6 fold higher than cytoplasmic variants ( cr-Hirudin and crhis-Hirudin). The prothrombin time and activated partial thromboplastin time of crhis-Hirudin and prhis-Hirudin were similar and approximately 20 and 55 % lower than those of cr-Hirudin.Conclusion: Applying signal peptide or His-tag increased the production yield of recombinant hirudin but had negative contributions to its activity.
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2. Tertrin C, De La Llosa P, Jutisz M. Effect of chemical modification of hirudin on its inhibition of the enzymatic activity of thrombin. Bull Soc Chim Biol 1967; 49(12):1837.
3. Markwardt F. Hirudin; an inhibitor of blood coagulation from medical leeches. Blut 1958; 4(3):160.
4. Landmann H. Hirudin and other thrombin inhibitors from blood-sucking animals. Folia Haematol (Leipz) 1972; 98(4):437.
5. Hallak O, Shams SA, Broce M, et al. Similar success rates with bivalirudin and unfractionated heparin in bare-metal stent implantation. Cardiovasc Intervent Radiol 2007; 30(5):906-11.
6. Herman K, Vaszily M, Szentgyörgyi L, et al. Anticoagulation with hirudin for repeat cardiac surgery in a patient with heparin induced thrombocytopaenia. Magy Seb 2008; 61:67-70.
7. Mureebe L. Direct thrombin inhibitors: alternatives to heparin. Vascular 2007;15(6):372-5.
8. Corral-Rodríguez MaAn, Macedo-Ribeiro S, Barbosa Pereira PJ, et al. Leech-derived thrombin inhibitors: from structures to mechanisms to clinical applications. J Med Chem 2010; 53(10):3847-61.
9. Markwardt F. Hirudin; an inhibitor of blood coagulation from medical leeches. Blut 1958; 4(3):160-1.
10. Piascik P. Medicinal leeches: ancient therapy is a source of biotech drugs. J Am Pharm Assoc 1997; 3:285-6.
11. Khoo KM, Chang CF, Schubert J, et al. Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system. Protein Expr Purif 2005; 40(2):396-403.
12. Croci C, Brandstätter JH, Enz R. ZIP3, a new splice variant of the PKC-ζ-interacting protein family, binds to GABAC receptors, PKC-ζ, and Kvβ2. J Biol Chem 2003; 278(8):6128-35.
13. Hochuli E, Döbeli H, Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 1987; 411:177-84.
14. Mason AB, He Q-Y, Halbrooks PJ, et al. Differential effect of a His tag at the N-and C-termini: functional studies with recombinant human serum transferrin. Biochemistry 2002; 41(30):9448-54.
15. Chant A, Provatopoulou X, Manfield IW, et al. Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA. Biochim Biophys Acta -Proteins Proteom - 2003; 1648(1-2):84-9.
16. Blobel G, Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 1975; 67(3):835-51.
17. Milstein C, Brownlee G, Harrison TM, et al. A possible precursor of immunoglobulin light chains. Nature New Biol 1972; 239(91):117.
18. Lee H, Chi S-W, Kang M, et al. Stability and folding of precursor and mature tryptophan-substituted ribose binding protein of Escherichia coli. Arch Biochem Biophys 1996; 328(1):78-84.
19. Goder V, Beckert V, Pfeil W, et al. Impact of the Presequence of a Mitochondrium-Targeted Precursor, Preadrenodoxin, on Folding, Catalytic Activity, and Stability of the Proteinin Vitro. Arch Biochem Biophys 1998; 359(1):31-41.
20. Asgari S, Mirzahoseini H, Karimipour M, et al. Rational design of stable and functional hirudin III mutants with lower antigenicity. Biologicals 2015; 43(6):479-91.
21. Reichelt P, Schwarz C, Donzeau M. Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expr Purif 2006; 46(2):483-8.
22. Asgari S, Ebrahim‐Habibi A, Mahdavi M, et al. Therapeutic protein deimmunization by T‐cell epitope removal: antigen‐specific immune responses in vitro and in vivo. Acta Pathol Microbiol Immunol Scand 2017; 125(6):544-52.
23. Greinacher A, Warkentin TE. The direct thrombin inhibitor Hirudin. Thromb Haemost 2008; 100(05):819-29.
24. Maraganore JM, Chao B, Joseph ML, et al. Anticoagulant activity of synthetic Hirudin peptides. J Biol Chem 1989; 264(15):8692-8.
25. Kobsar A, Koessler J, Kehrer L, et al. The thrombin inhibitors hirudin and Refludan activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets. Thromb Haemost 2012; 107(03):521-9.
26. Pitt B. Cardiovascular Therapeutics: A Companion to Braunwald's heart disease. Circulation 1997; 96(3):1041-2.
27. Liu G, Topping TB, Cover WH, et al Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein. J Biol Chem 1988;263(29):14790-3.
28. Diamond DL, Strobel S, Chun SY, et al. Interaction of SecB with intermediates along the folding pathway of maltose‐binding protein. Protein Sci 1995; 4(6):1118-23.
29. Raffy S, Sassoon N, Hofnung M, et al. Tertiary structure‐dependence of misfolding substitutions in loops of the maltose‐binding protein. Protein Sci 1998; 7(10):2136-42.
30. Chun S-Y, Strobel S, Bassford P, et al. Folding of maltose-binding protein. Evidence for the identity of the rate-determining step in vivo and in vitro. J Biol Chem 1993; 268(28):20855-62.
31. Beena K, Udgaonkar JB, Varadarajan R. Effect of signal peptide on the stability and folding kinetics of maltose binding protein. Biochemistry 2004; 43(12):3608-19.
32. Porath J, Carlsson J, Olsson I, et al. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975; 258(5536):598.
33. Gräslund S, Nordlund P, Weigelt J, et al. Protein production and purification. Nat Methods 2008; 5(2):135.
34. Nilsson B, Forsberg G, Moks T, et al. Fusion proteins in biotechnology and structural biology. Curr Opin Struct Biol 1992; 2(4):569-75.
35. Majorek KA, Kuhn ML, Chruszcz M, et al. Double trouble—Buffer selection and H is‐tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci 2014; 23(10):1359-68.
36. Deller MC, Kong L, Rupp B. Protein stability: a crystallographer's perspective. Acta Crystallogr Sect F: Struct Biol Commun 2016; 72(2):72-95.
37. Zhao D, Huang Z. Effect of his-tag on expression, purification, and structure of zinc finger protein, ZNF191 (243-368). Bioinorg Chem Appl 2016;2016: 1-6.
2. Tertrin C, De La Llosa P, Jutisz M. Effect of chemical modification of hirudin on its inhibition of the enzymatic activity of thrombin. Bull Soc Chim Biol 1967; 49(12):1837.
3. Markwardt F. Hirudin; an inhibitor of blood coagulation from medical leeches. Blut 1958; 4(3):160.
4. Landmann H. Hirudin and other thrombin inhibitors from blood-sucking animals. Folia Haematol (Leipz) 1972; 98(4):437.
5. Hallak O, Shams SA, Broce M, et al. Similar success rates with bivalirudin and unfractionated heparin in bare-metal stent implantation. Cardiovasc Intervent Radiol 2007; 30(5):906-11.
6. Herman K, Vaszily M, Szentgyörgyi L, et al. Anticoagulation with hirudin for repeat cardiac surgery in a patient with heparin induced thrombocytopaenia. Magy Seb 2008; 61:67-70.
7. Mureebe L. Direct thrombin inhibitors: alternatives to heparin. Vascular 2007;15(6):372-5.
8. Corral-Rodríguez MaAn, Macedo-Ribeiro S, Barbosa Pereira PJ, et al. Leech-derived thrombin inhibitors: from structures to mechanisms to clinical applications. J Med Chem 2010; 53(10):3847-61.
9. Markwardt F. Hirudin; an inhibitor of blood coagulation from medical leeches. Blut 1958; 4(3):160-1.
10. Piascik P. Medicinal leeches: ancient therapy is a source of biotech drugs. J Am Pharm Assoc 1997; 3:285-6.
11. Khoo KM, Chang CF, Schubert J, et al. Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system. Protein Expr Purif 2005; 40(2):396-403.
12. Croci C, Brandstätter JH, Enz R. ZIP3, a new splice variant of the PKC-ζ-interacting protein family, binds to GABAC receptors, PKC-ζ, and Kvβ2. J Biol Chem 2003; 278(8):6128-35.
13. Hochuli E, Döbeli H, Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 1987; 411:177-84.
14. Mason AB, He Q-Y, Halbrooks PJ, et al. Differential effect of a His tag at the N-and C-termini: functional studies with recombinant human serum transferrin. Biochemistry 2002; 41(30):9448-54.
15. Chant A, Provatopoulou X, Manfield IW, et al. Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA. Biochim Biophys Acta -Proteins Proteom - 2003; 1648(1-2):84-9.
16. Blobel G, Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 1975; 67(3):835-51.
17. Milstein C, Brownlee G, Harrison TM, et al. A possible precursor of immunoglobulin light chains. Nature New Biol 1972; 239(91):117.
18. Lee H, Chi S-W, Kang M, et al. Stability and folding of precursor and mature tryptophan-substituted ribose binding protein of Escherichia coli. Arch Biochem Biophys 1996; 328(1):78-84.
19. Goder V, Beckert V, Pfeil W, et al. Impact of the Presequence of a Mitochondrium-Targeted Precursor, Preadrenodoxin, on Folding, Catalytic Activity, and Stability of the Proteinin Vitro. Arch Biochem Biophys 1998; 359(1):31-41.
20. Asgari S, Mirzahoseini H, Karimipour M, et al. Rational design of stable and functional hirudin III mutants with lower antigenicity. Biologicals 2015; 43(6):479-91.
21. Reichelt P, Schwarz C, Donzeau M. Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expr Purif 2006; 46(2):483-8.
22. Asgari S, Ebrahim‐Habibi A, Mahdavi M, et al. Therapeutic protein deimmunization by T‐cell epitope removal: antigen‐specific immune responses in vitro and in vivo. Acta Pathol Microbiol Immunol Scand 2017; 125(6):544-52.
23. Greinacher A, Warkentin TE. The direct thrombin inhibitor Hirudin. Thromb Haemost 2008; 100(05):819-29.
24. Maraganore JM, Chao B, Joseph ML, et al. Anticoagulant activity of synthetic Hirudin peptides. J Biol Chem 1989; 264(15):8692-8.
25. Kobsar A, Koessler J, Kehrer L, et al. The thrombin inhibitors hirudin and Refludan activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets. Thromb Haemost 2012; 107(03):521-9.
26. Pitt B. Cardiovascular Therapeutics: A Companion to Braunwald's heart disease. Circulation 1997; 96(3):1041-2.
27. Liu G, Topping TB, Cover WH, et al Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein. J Biol Chem 1988;263(29):14790-3.
28. Diamond DL, Strobel S, Chun SY, et al. Interaction of SecB with intermediates along the folding pathway of maltose‐binding protein. Protein Sci 1995; 4(6):1118-23.
29. Raffy S, Sassoon N, Hofnung M, et al. Tertiary structure‐dependence of misfolding substitutions in loops of the maltose‐binding protein. Protein Sci 1998; 7(10):2136-42.
30. Chun S-Y, Strobel S, Bassford P, et al. Folding of maltose-binding protein. Evidence for the identity of the rate-determining step in vivo and in vitro. J Biol Chem 1993; 268(28):20855-62.
31. Beena K, Udgaonkar JB, Varadarajan R. Effect of signal peptide on the stability and folding kinetics of maltose binding protein. Biochemistry 2004; 43(12):3608-19.
32. Porath J, Carlsson J, Olsson I, et al. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975; 258(5536):598.
33. Gräslund S, Nordlund P, Weigelt J, et al. Protein production and purification. Nat Methods 2008; 5(2):135.
34. Nilsson B, Forsberg G, Moks T, et al. Fusion proteins in biotechnology and structural biology. Curr Opin Struct Biol 1992; 2(4):569-75.
35. Majorek KA, Kuhn ML, Chruszcz M, et al. Double trouble—Buffer selection and H is‐tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci 2014; 23(10):1359-68.
36. Deller MC, Kong L, Rupp B. Protein stability: a crystallographer's perspective. Acta Crystallogr Sect F: Struct Biol Commun 2016; 72(2):72-95.
37. Zhao D, Huang Z. Effect of his-tag on expression, purification, and structure of zinc finger protein, ZNF191 (243-368). Bioinorg Chem Appl 2016;2016: 1-6.
| Files | ||
| Issue | Vol 10 No 1-2 (2021) | |
| Section | Original Articles | |
| Keywords | ||
| Hirudin His-tag signal peptide PT assay PTT assay | ||
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How to Cite
1.
Hadadian S, Pishkar L, kazemi E, Asgari S, Sepahi M, Komijani S, Afrough P, Mohajerani N. The Effect of Signal Sequence and His-tags on the Expression and Anti-Thrombin Activity of Recombinant Hirudin. J Med Bacteriol. 2021;10(1,2):11-19.
