بیان پروتئین آلفا-لاکتالبومین انسانی با استفاده از سازه کلروپلاستی در باکتری Escherichia coli

نوع مقاله : مقاله پژوهشی

نویسندگان

گروه بهنژادی و بیوتکنولوژی گیاهی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران

چکیده

هدف: با افزایش تقاضای جهانی برای منابع پروتئینی با کیفیت بالا، تولید نوترکیب پروتئین‌های شیر انسانی به‌عنوان گزینه‌ای ایمن، مغذی و پایدار مورد توجه قرار گرفته است. این پژوهش با هدف طراحی و ارزیابی اولیه یک کاست بیانی کلروپلاستی حاوی ژن آلفا-لاکتالبومین انسانی در باکتری Escherichia coli به‌عنوان گام مقدماتی در مسیر تولید گیاهی این پروتئین انجام شد.
مواد و روش‌ها: توالی رمزکننده آلفا-لاکتالبومین انسانی با کدون‌های ترجیحی کلروپلاست توتون بهینه‌سازی و در قالب یک کاست بیانی شامل راه‌انداز rrn، ناحیه 5′UTR ژن 10 فاژ 7T و پایان‌دهنده rrnB طراحی گردید. این سازه در ناقل کلروپلاستی pPRV111A همسانه‌سازی شد و با استفاده از واکنش زنجیره‌ای پلیمراز و برش آنزیمی، صحت ساختاری آن تأیید شد. جهت ارزیابی کارایی ترجمه، پلاسمید نوترکیب به E. coli سویه BL21 (DE3) منتقل و القای بیان با IPTG انجام شد. پروتئین حاصل با روش کروماتوگرافی تمایلی بر پایه رزین نیکل تخلیص و پس از حذف برچسب هیستیدینی با آنزیم TEV، مجموعه HAMLET با اسید اولئیک تولید گردید. فعالیت زیستی این مجموعه با آزمون MTT روی رده سلولی WiDr ارزیابی شد.
نتایج: پروتئین آلفا-لاکتالبومین انسانی با وزن تقریبی 16 کیلو دالتون به‌صورت محلول در باکتری بیان شد. خالص‌سازی مؤثر پروتئین از بخش محلول سلولی و حذف موفق برچسب هیستیدینی، منجر به تولید پروتئینی با وزن نهایی حدود 14 کیلو دالتون شد. آزمون‌های تأییدی شامل لکه‌گذاری وسترن و SDS-PAGE بیان موفق و خلوص بالای پروتئین را تأیید کردند. مجموعه HAMLET تهیه‌شده با ترکیب این پروتئین و اسید اولئیک، در آزمون MTT موجب کاهش قابل‌توجه زنده‌مانی رده سلولی WiDr شد. به‌طور خاص، تیمار سلول‌ها با 21 میکرومولار HAMLET باعث کاهش 96 درصدی بقاء سلولی گردید، در حالی‌که تیمارهای جداگانه با آلفا-لاکتالبومین یا اسید اولئیک فاقد اثر معنی‌دار بودند. این نتایج نشان دادند که پروتئین تولیدشده نه‌تنها از نظر ساختاری صحیح است، بلکه ویژگی‌های عملکردی و زیستی مورد انتظار خود را نیز حفظ کرده است.
نتیجه‌گیری: کاست بیانی طراحی‌شده دارای کارایی بالا در سیستم باکتریایی بوده و توانسته پروتئینی با فعالیت زیستی مطلوب تولید کند. این مطالعه شواهدی تجربی برای امکان‌سنجی استفاده از این سازه در تراریختی کلروپلاست گیاهان فراهم کرده و گامی مؤثر در مسیر تولید صنعتی آلفا-لاکتالبومین انسانی و ترکیبات زیست‌فعال مشتق از آن مانند HAMLET محسوب می‌شود.

کلیدواژه‌ها

عنوان مقاله [English]

Expression of human alpha-lactalbumin using a chloroplast-based construct in Escherichia coli

نویسندگان [English]

  • Maryam Ehsasatvatan
  • Bahram Baghban Kohnehrouz
Department of Plant Breeding & Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
چکیده [English]

Objective
With the increasing global demand for high-quality protein sources, the recombinant production of human milk proteins has gained attention as a safe, nutritious, and sustainable alternative. This study aimed to design and perform a preliminary evaluation of a chloroplast-compatible expression cassette containing the human α-lactalbumin gene in Escherichia coli, as an initial step toward plant-based production of this protein.
Materials and methods
The coding sequence of human α-lactalbumin was codon-optimized based on the preferred codon usage of tobacco chloroplasts and assembled into an expression cassette consisting of the rrn promoter, the 5′ untranslated region (UTR) of T7 phage gene 10, and the rrnB terminator. The cassette was cloned into the chloroplast vector pPRV111A and validated by polymerase chain reaction and restriction enzyme analysis. Translational efficiency was assessed by transformation of the recombinant plasmid into E. coli strain BL21 (DE3) and protein expression induction using IPTG. The recombinant protein was purified via nickel-based affinity chromatography, followed by His-tag removal using TEV protease. The HAMLET complex was formed by combining the purified protein with oleic acid, and its biological activity was assessed using the MTT assay on WiDr colorectal cancer cells.
Results
Human α-lactalbumin was successfully expressed in in E. coli in soluble form, with an approximate molecular weight of 16 kDa. Efficient purification from the soluble protein fraction and successful removal of the His-tag yielded a final product of ~14 kDa. Western blotting and SDS-PAGE verified successful expression and high protein purity. The HAMLET complex formed by combining the purified protein with oleic acid significantly reduced the viability of WiDr cells in the MTT assay. Specifically, treatment with 21 µM HAMLET resulted in a 96% decrease in cell survival, whereas treatments with α-lactalbumin or oleic acid alone had no significant effect. These findings demonstrate that the expressed protein retained both correct structural integrity and expected biofunctional properties.
Conclusions
The designed expression cassette showed high efficiency in the bacterial system and enabled the production of biologically active human α-lactalbumin. This study provides experimental evidence supporting the feasibility of using the designed construct for chloroplast transformation in plants, representing a promising step toward the industrial production of recombinant α-lactalbumin and its bioactive derivatives such as HAMLET.

کلیدواژه‌ها [English]

  • Human α-lactalbumin
  • Bacterial expression
  • Whey protein
  • Chloroplastic vector

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مجله بیوتکنولوژی کشاورزی
دوره 18، شماره 1
فروردین 1405
صفحه 77-96

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