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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آمار تعداد مشاهده مقاله: 929 تعداد دریافت فایل اصل مقاله: 860 |
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