بهینه‌سازی شرایط تخلیص و بازآرایی پروتئین نوترکیب اندونوکلئاز محدودکننده EcoRI در E.coli

نوع مقاله : مقاله پژوهشی

نویسندگان

1 استادیار، پژوهشکده ژنتیک و زیست فناوری طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران.

2 دکتری، پژوهشکده ژنتیک و زیست فناوری طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران.

3 استاد، پژوهشکده ژنتیک و زیست فناوری طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

چکیده

هدف: اندونوکلئازهای محدودکننده نوع دوم، متداول‌ترین نوع آنزیم‌های محدودکننده هستند که کاربرد وسیعی در مهندسی ژنتیک به‌ویژه همسانه‌سازی ژن‌ها دارند. آنزیم EcoRI از پرمصرف‌ترین آنزیم‌های محدودکننده نوع دو در حوزه مولکولی است. روش مرسوم تولید این آنزیم‌ها استفاده از سویه‌های تغییریافتة باکتری Escherichia coli است که به‌منظور بیش تولید آنزیم EcoRI جهش یافته‌اند. خالص‌سازی آنزیم‌های تولید شده با این روش به ستون‌های متعدد کروماتوگرافی نیاز دارد که هزینه بر و وقت‌گیر است. باتوجه‌به اینکه آنزیم EcoRI دارای ساختاری ساده و بدون پل‌های دی سولفید و به‌صورت مونومر است در این مطالعه تولید آنزیم به روش نوترکیب، روش بهینه خالص‌سازی و بازآرایی این آنزیم مورد بررسی قرار گرفت.
مواد و روش:ژن EcoRI از باکتری اشرشیا کولای (E. coli RY13) جداسازی گردید و به‌منظور بیان ژن به‌صورت نوترکیب، در حامل بیانی pET28 همسانه‌سازی و در باکتری E. coli BL21 (DE3) بیان شد. شرایط بهینه بیان پروتئین باتوجه‌به نوع القاگر، غلظت و دما پس از القا مورد بررسی قرار گرفت. بیان پروتئین به‌صورت محلول یا اجسام انکلوزیونی با استفاده از ژل SDS-PAGE بررسی شد. خالص‌سازی و بازآرایی پروتئین نوترکیب با دو روش کروماتوگرافی تمایلی و دیالیز انجام شد. فعالیت آنزیم در مقایسه با آنزیم تجاری EcoRI شرکت ترمو بررسی شد.
نتایج: شرایط بهینه بیان EcoRI در غلظت ۸/۰ میلی‌مولار IPTG، دمای ۲۸ درجه سانتی‌گراد تعیین شد. نتایج حاصل از بررسی پروتئین نوترکیب بیان شده روی ژل SDS-PAGE نشان داده است که پروتئین نوترکیب به شکل اجسام انکلوزیونی بیان گردید همچنین حل‌شدن اجسام انکلوزیونی تحت شرایط ملایم واسرشته‌‌سازی اوره ۳ مولار نشان داد که اجسام انکلوزیونی از نوع غیرکلاسیک است. نتایج حاصل از بازآرایی اجسام انکلوزیونی با استفاده از دیالیز و بر روی رزین Ni-NTA مشخص کرد که بازدهی بازآرایی بر روی رزین بیشتر از بازآرایی با استفاده از دیالیز است. واکنش هضم آنزیمی با آنزیم تولیدشده و آنزیم تجاری نشان داد که آنزیم نوترکیب خالص‌شده مشابه آنزیم تجاری قادر به برش DNA پلاسمیدی است.
نتیجه‌گیری: بازآرایی اجسام انکلوزیونی بر روی ستون منجر به صرفه‌جویی در وقت و هزینه شده و کارایی بیشتری نسبت به روش دیالیز دارد.

کلیدواژه‌ها

عنوان مقاله [English]

Optimizing purification and refolding conditions for the recombinant EcoRI restriction endonuclease protein in E. coli

نویسندگان [English]

  • Camellia Katalani 1
  • Elham Soleimani 2
  • Ghorbanali Nematzadeh 3
1 Assistant Professor, Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran.
2 Ph.D. Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran.
3 Professor, Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran.
چکیده [English]

Objective: Type II restriction endonucleases are the most common type of restriction enzymes, widely used in genetic engineering, particularly in gene cloning. Among these enzymes, the EcoRI enzyme is one of the most frequently utilized in the molecular field. The conventional method for producing these enzymes involves using genetically modified strains of Escherichia coli, which have been mutated to overproduce the EcoRI enzyme. However, the purification process for these enzymes is both costly and time-consuming, as it requires multiple chromatography columns. Given that the EcoRI enzyme has a simple structure without disulfide bridges and exists as a monomer, this study investigates the recombinant production, optimized purification, and refolding of this enzyme.
Material and methods: The EcoRI gene was isolated from the bacterium Escherichia coli (E. coli RY13) and cloned into the expression vector pET28 for recombinant expression in E. coli BL21 (DE3). The optimal conditions for protein expression were investigated, including the type of inducer,its concentration, and the temperature after induction. Protein expression was evaluated in terms of soluble protein or inclusion bodies using SDS-PAGE gel analysis. The recombinant protein was then purified and refolded using affinity chromatography and dialysis methods. Finally, the enzyme activity was compared to that of the commercial EcoRI enzyme from Thermo
Results: The optimal conditions for the expression of the EcoRI were determined to be 0.8 mM IPTG concentration and a temperature of 28°C. The results of the analysis of the recombinant protein expressed on SDS-PAGE gel indicated that the EcoRI was expressed as inclusion bodies. The solubilization of these inclusion bodies under mild conditions and refolding with 3 M urea demonstrated that the inclusion bodies were of a non-classical type. Furthermore, results from the refolding process, using both dialysis and Ni-NTA column, showed that the yield of refolding on the resin was higher than that obtained through dialysis. Enzymatic digestion reactions using the produced enzyme and a commercial enzyme indicated that the purified recombinant enzyme was capable of cutting plasmid DNA, similar to the commercial enzyme.
Conclusion: Refolding of inclusion bodies on the column resulted in significant time and cost savings, demonstrating greater efficiency compared to the dialysis method.

کلیدواژه‌ها [English]

  • EcoRI enzyme
  • inclusion body
  • protein refolding
  • protein purification

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مجله بیوتکنولوژی کشاورزی
دوره 17، شماره 2
اردیبهشت 1404
صفحه 107-132

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