Production of mutant streptavidin protein and investigation of its effect on the performance of streptavidin | ||
| Iranian Journal of Basic Medical Sciences | ||
| مقاله 11، دوره 26، شماره 5، مرداد 2023، صفحه 572-578 اصل مقاله (681.49 K) | ||
| نوع مقاله: Original Article | ||
| شناسه دیجیتال (DOI): 10.22038/ijbms.2023.69083.15060 | ||
| نویسندگان | ||
| Elham Didevara؛ Shabnam Sadoogh Abbassian؛ Abdolrahime Sadeghi؛ Hamid Abtahi* | ||
| Molecular and Medical Research Center, Arak University of Medical Sciences, Arak, Iran | ||
| چکیده | ||
| Objective(s): Streptavidin is a versatile protein in cell science. The tetramer structure of streptavidin plays a key role in this binding, but this form interferes with some assays. If monomer streptavidin is still capable of binding to biotin, it can overcome the limitations of the streptavidin application. So, we examined the elimination of tryptophan 120 and its effect on the function of streptavidin. Materials and Methods: Mutant streptavidin gene was synthesized in a pBSK vector. Then it was ligated to the pET32α vector. This vector is expressed in Escherichia coli BL21 (DE3) pLysS host. After purification and refolding of the recombinant protein, its structure was analyzed on the SDS_PAGE gel. Recombinant streptavidin binding affinity to biotin was evaluated by spectrophotometric and HABA color compound. Results: Mutant streptavidin gene was successfully expressed in E. coli BL21 (DE3) pLysS host and the purified protein was observed as a single band in the 36 kDa area. The best condition for dialysis was PBS buffer+arginine. The molar ratio of biotin/protein of mutant streptavidin was not only near but also more than standard protein. Mutant streptavidin remained in the monomeric state in the presence or absence of biotin. Conclusion: Results of this study showed that 120 tryptophan is one of the most important factors in tetramer streptavidin formation and its deletion produces the monomer form that has a high binding affinity to biotin. This mutant form of streptavidin can therefore be used in studies requiring monovalent binding as well as in studies facing limitations due to the size of streptavidin tetramer. | ||
| کلیدواژهها | ||
| Arginine؛ Biotin؛ HABA؛ Recombinant Proteins؛ Streptavidin | ||
| مراجع | ||
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1. Laitinen OH, Hytönen VP, Nordlund HR, Kulomaa MS. Genetically engineered avidins and streptavidins. Cell Mol Life Sci 2006 ;63:2992-3017. 2. Chivers CE, Koner AL, Lowe ED, Howarth M. How the biotin-streptavidin interaction was made even stronger: Investigation via crystallogra-phy and a chimaeric tetramer. Biochem J 2011; 435: 55-63. 3. Dundas CM, Demonte D, Park S. Streptavidin–biotin technology: Improvements and innovations in chemical and biological applications. Appl Microbiol Biotechnol 2013;97:9343-9353. 4. Yanai I, Yu Y, Zhu X, Cantor CR, Weng Z. An avidin-like domain that does not bind biotin is adopted for oligomerization by the extracellular mosaic protein fibropellin. Protein Sci 2005;14:417-423. 5. Futch L, Pham T, Adamson B, Reilly B, Kronenfeld J, Patterson K, et al. It’s Strong, It’s Stable, It’s Streptavidin. FASEB J 2015;29:LB57. 6. Molaee N, Abtahi H, Mosayebi G. Expression of recombinant streptokinase from Streptococcus pyogenes and its reaction with infected human and murine sera. Iran J Basic Med Sci 2013;16, 985-989. 7. Hasanzadeh L, Ghaznavi-Rad E, Soufian S, Farjadi V, Abtahi H. Expression and antigenic evaluation of VacA antigenic fragment of Helicobacter pylori. Iran J Basic Med Sci 2013; 16: 835–840. 8. Abtahi H, Salmanian A.H, Rafati S, Nejad G.B, Hassan Z.M. High level expression of recombinant ribosomal protein (L7/L12) from Brucella abortus and its reaction with infected human sera. Iran Biomed J 2004; 8:13–18. 9. Mirjamali N, Soufian, S, Molaee N, Sadoogh Abbasian S, Abtahi H. Cloning and expression of the enzymatic region of Streptococcal hyaluronidase. Iran J Basic Med Sci 2014; 17:667–672. 10. Farjadi V, Abtahi H, Zolfaghari M.R, Soufian S, Hasanzadeh L. Expression, purification and evaluation of antigenicity of CagA antigenic fragment of Helicobacter pylori. Jundishapur J Microbiol 2013; 6:e7367. 11. Silva FSR, Santos SPO, Meyer R, Silva ES, Pinheiro CS, Alcantara-Neves NM, Pacheco LGC. In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli. Biotechnol Bioeng 2021;118:4159-4167. 12. Han SH, Kim HM, Lim MW, Kim J-K. Functional Expression of Soluble Streptavidin in Escherichia coli. J Life Sci 2015; 25:631-637. 13. Laitinen O, Hytönen V, Nordlund H, Kulomaa M. Genetically engineered avidins and streptavidins. Cell Mol Life Sci 2006;63:2992-3017. 14. Qureshi MH, Yeung JC, Wu SC, Wong SL. Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities. J Biol Chem 2001;7;276:46422-46428. 15. Arakawa T, Ejima D, Tsumoto K, Obeyama N, Tanaka Y, Kita Y, et al. Suppression of protein interactions by arginine: A proposed mechanism of the arginine effects. Biophys Chem 2007;127:1-8. 16. Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat Methods 2006;3:267-273. 17. Baugh L, Le Trong I, Cerutti DS, Gülich S, Stayton PS, Stenkamp RE, Lybrand TP. A distal point mutation in the streptavidin-biotin complex preserves structure but diminishes binding affinity: Experimental evidence of electronic polarization effects? Biochemistry 2010; 8;49:4568-4570. 18. Demonte D, Dundas CM, Park S. Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl Microbiol Biotechnol 2014;98:6285-6295. 19. Kroetsch A, Chin B, Nguyen V, Gao J, Park S. Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: Applications in flow cytometry and ELISA. Appl Microbiol Biotechnol 2018;102:10079-10089. 20. Chamma I, Rossier O, Lim KH, Gauthereau I, Giannone G, Park S, et al. Super resolution mapping of adhesion molecules in confined cellular environments using monomeric streptavidin ligands. Biophys J 2014;106:202a. 21. Jurchenko C, Chang Y, Narui Y, Zhang Y, Salaita KS. Integrin-generated forces lead to streptavidin-biotin unbinding in cellular adhesions. Biophys J 2014;1;106:1436-1446. 22. Lim KH, Huang H, Pralle A, Park S. Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol Bioeng 2013;110:57-67. 23. Lim KH, Huang H, Pralle A, Park S. Engineered streptavidin monomer and dimer with improved stability and function. Biochemistry 2011;11;50:8682-8691. 24. Wu SC, Wong SL. Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem 2005;280:23225-23231. 25. O’Sullivan VJ, Barrette-Ng I, Hommema E, Hermanson GT, Schofield M, Wu SC, et al. Development of a tetrameric streptavidin mutein with reversible biotin binding capability: Engineering a mobile loop as an exit door for biotin. PLoS One 2012;7:e35203. | ||
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