یافته های نوین در علوم زیستی

بهینه سازی تخلیص پروتئین لایه S از سویه Deinococcus radiodurans R1

نویسندگان

مهدی علی جانیان زاده 1
علیرضا جلالوند 2
رسول خلیل زاده 3
مریم عبدلی راد 3

1 گروه علوم سلولی مولکولی، دانشکده علوم زیستی، دانشگاه خوارزمی، کرج، ایران

2 بخش آنفولانزا و ویروس‌های تنفسی شایع، انستیتو پاستور ایران، تهران، ایران

3 پژوهشکده بیوتکنولوژی، دانشگاه صنعتی مالک اشتر، تهران، ایران

چکیده
پروتئین‌های لایه سطحی Deinococcus radiodurans یکی از بهترین سیستم های خودآرایی در بین پروتئین‌های دیگر هستند که نقش اساسی در ساخت نانوسیم ها دارند. بنابراین لازم است این پروتئین‌ها خالص سازی شوند. هدف از این تحقیق بهینه سازی خالص سازی پروتئین لایه سطحی از D. radiodurans با روش سطح پاسخ است. ابتدا سه عامل غلظت SDS، زمان انکوباسیون و درصد جرم در پنج سطح در نظر گرفته شد و 20 اجرا با نرم افزار Design-Expert با روش مرکب مرکزی طراحی شد. هر مرحله شامل کشت میکروب، آماده سازی سلول انبوه، انکوباسیون میکروب در غلظت خاص SDS و زمان و درصد جرم، جداسازی باکتری از مواد شوینده با سانتریفیوژ در g5000، ته نشینی پروتئین‌های لایه سطحی از محلول شوینده با سانتریفیوژ در g20000، تعیین غلظت و خلوص پروتئین به ترتیب با روش برادفورد و SDS-PAGE است. در نهایت داده‌های به دست آمده مورد تجزیه و تحلیل قرار گرفت. تجزیه و تحلیل نتایج نشان داد که در سطح اطمینان 95 درصد، تأثیر فاکتور غلظت ماده شوینده بر درصد پروتئین خالص شده بیشتر از سایر عوامل بود. نتایج بهینه سازی فاکتورها بدین صورت بود: غلظت SDS 64/5 درصد، درصد جرم 7/33 درصد و 3 ساعت زمان انکوباسیون. در شرایط بهینه، غلظت پروتئین و درصد خلوص به ترتیب mg/ml 0/584 و 47/61 درصد به دست آمد.


کلیدواژه‌ها

الکتروفورز
پروتئین لایه سطحی
تخلیص
سطح پاسخ
شوینده

عنوان مقاله English

Optimization of S-layer protein purification from Deinococcus radiodurans strain R1

نویسندگان English

Mahdi Alijanianzadeh 1
Alireza Jalalvand 2
Rasoul Khalilzadeh 3
Maryam Abdolirad 3
1 Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Karaj, Iran
2 Department of Influenza and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran
3 Department of Bioscience and Biotechnology, Malek-Ashtar University of Technology, Tehran, Iran
چکیده English

S-layer proteins of Deinococcus radiodurans are the best self-assemble systems among other proteins that have an essential role in the fabrication of nanowires. Therefore, the purification of these proteins is necessary. The purpose of this research was to optimize the purification of s-layer protein from D. radiodurans with the response surface method. The three factors of SDS concentration, incubation time and mass percent in five levels were considered, and 20 runs were designed by Design-Expert software with a central composite method. Each run includes microbe culture, mass cell preparation, microbe incubation in specific SDS concentration and time and mass percent, separation of the bacteria from detergent with a centrifuge at 5000g, sedimentation of s-layer proteins from detergent solution with a centrifuge at 20000g, determination of protein concentration, and protein purity by Bradford and SDS-PAGE methods, respectively. Finally, the data obtained were analyzed. Analysis of the results demonstrated that at the 95% confidence level, the effect of the detergent concentration factor on the purified protein percent was more than other factors. The optimization results of factors are 5.64% SDS concentration, 7.33% mass percent, and 3 hours incubation time. At optimized conditions the protein concentration and purity percent were obtained 0.584 mg/ml and 47.61% respectively.


کلیدواژه‌ها English

Electrophoresis
detergent
purification
response surface
surface layer protein
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  • تاریخ دریافت 18 خرداد 1405
  • تاریخ انتشار 18 خرداد 1405

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