یافته های نوین در علوم زیستی

بیان هم‌زمان فاکتور رشد عصبی نوترکیب انسانی با چاپرون تریگر فاکتور در E. coli

نویسندگان

سیده مهدیه سادات
زهرا حاجی حسن
محمد برشان تشنیزی
مهری عبدی

گروه مهندسی علوم زیستی، دانشکده علوم و فنون نوین، دانشگاه تهران

https://doi.org/10.29252/nbr.5.3.221
چکیده
. فاکتور رشد عصبی (NGF) یک فاکتور نوروتروفیک است که در حفظ، بقا و تمایز سلول‌های سیستم عصبی نقش دارد. پروتئین NGF دارای سه زیر واحد است که زیر واحد β آن فعالیت اصلی را بر عهده دارد. این پروتئین از موتیف گره سیستئینی که در آن رشته‌های β با پیوندهای دی سولفیدی به یکدیگر متصل شده‌اند، تشکیل ‌شده است. NGF برای درمان بسیاری از بیماری‌ها استفاده می‌شود. به دلیل اینکه NGF استخراج ‌شده از منابع طبیعی برای درمان نامناسب است، بسیاری از محققین برای تولید – NGFβ نوترکیب تلاش کرده‌اند. در این مطالعه، به ‌منظور افزایش بیان NGF، این پروتئین به ‌طور هم ‌زمان با چپرون Trigger Factor در E. coli بیان شد. بدین منظور pET39b(+)::β-NGF و پلاسمید چپرونی pTF16 به E. coli BL21(DE3) انتقال یافتند. پس از القای هر پروموتر، کل محتوی پروتئینی و پروتئین های پری پلاسمی بطور جداگانه استخراج شدند. به منظور تأیید تأثیر TFبر میزان بیان کلی و تولید –NGFβ محلول، تکنیک‌های برادفورد و دات بلات و نرم‌افزار ImageJ استفاده شدند. سپس –NGFβ با استفاده از ستون کروماتوگرافی تمایلی (Ni2+-NTA) تخلیص شد. همچنین به منظور مطالعه عملکرد NGF-β تخلیص شده، رده سلول‌های PC12 با پروتئین به مدت یک هفته تیمار شدند. داده‌ها نشان دادند که بیان هم‌زمان با چپرون TF می‌تواند سبب افزایش تولید پری پلاسمی و محلول –NGFβ و نه کل محتوی پروتئینی شود. همچنین تیمار سلول‌های PC12 با –NGFβ تخلیص شده (بیان‌شده با چپرون TF)، منجر به تمایز سلول های PC12 به سلول های عصبی شد، که نشان‌دهنده عملکردی بودن پروتئین تولید شده است. داده‌های این مطالعه نشان‌دهنده این است که بیان هم‌زمان چپرون سیتوپلاسمی TF با پروتئین NGF ممکن است یک روش مؤثر در تولید مناسب فاکتور رشد عصبی نوترکیب (rhNGF) محلول و فعال باشد.

کلیدواژه‌ها

بیان هم‌زمان
تولید پری پلاسمی
فاکتور راه انداز
فاکتور رشد عصبی
کروماتوگرافی تمایلی

عنوان مقاله English

Co-expression of recombinant human nerve growth factor with trigger factor chaperone in E. coli

نویسندگان English

Seyedeh Mahdieh Sadadt
Zahra Hajihassan
Mohammad Barshan-tashnizi
Mehri Abdi
University of Tehran,
چکیده English

Nerve growth factor (NGF) is a neurotrophic factor that is functional in the survival, maintenance and differentiation of nervous system cells. This protein has three subunits, of which the beta subunit has the main activity. Its structure consists of a cysteine knot motif made up of beta strands linked by disulfide bonds. It can be used as a therapeutic agent in the treatment of many diseases. As NGF extracted from natural sources is unsuitable for therapeutic goals, many studies have attempted to produce recombinant β-NGF. In this study, Trigger Factor (TF) chaperone was expressed simultaneously with β-NGF in E. coli in order to obtain increased yield of soluble recombinant human β-NGF. For this purpose, pET39b (+)::β-NGF and chaperone plasmid pTf16 were transferred to E.coli (DE3 strain). After the induction of each promoter, the total proteins and periplasmic proteins were extracted. To confirm the effects of TF on total protein and soluble β-NGF expression level, Bradford and Dot blot techniques and ImageJ software were used. Then, β-NGF was purified using affinity chromatography column (Ni+2-NTA). Also, the PC12 cells were treated with the protein for one week in order to study the function of purified NGF. Our data indicated that co-expression of TF could increase the soluble and periplasmic production of β-NGF but not total proteins. Also, the treatment of PC12 cell line with purified β-NGF, co-expressed with TF chaperone, showed differentiation of these cells to nerve cells. This indicated that the purified NGF is fully functional. Our data suggest that the co-expression of cytoplasmic chaperone (TF) with recombinant nerve growth factor might be an efficient approach to produce a proper quantity of soluble and active rhNGF.

کلیدواژه‌ها English

affinity chromatography
co-expression
Nerve Growth Factor
periplasmic production
trigger factor
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  • تاریخ دریافت 18 خرداد 1405
  • تاریخ انتشار 18 خرداد 1405

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