Design and Construction of Recombinant ELP-Intein Cassette for Use in Simple and new Purification Methods of Recombinant Proteins

Publish Year: 1397
نوع سند: مقاله ژورنالی
زبان: English
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شناسه ملی سند علمی:

JR_MML-1-2_006

تاریخ نمایه سازی: 25 اردیبهشت 1401

Abstract:

Background and Objective: Use of elastin-like proteins (ELPs) provides high-performance protein purification without need for chromatography. In line with cost reduction and facilitation of recombinant proteins purification, which represent a high percentage of production costs, in this project, we eliminated the need for proteases in the process of separation of recombinant proteins from ELP by designing a cassette using ELPs properties as well as insertion of autocatalytic intein protein between the recombinant protein and ELP. Methods: In this study, at first Mxe GyrA intein gene was amplified from pTXB۱ vector by PCR method and cloned into pUC۵۷-hEGF vector. Then, ۸xELP repetitive sequences were first cloned in pUC۵۷ vector and then into pUC۵۷-intein-hEGF vector in the upstream of Intein-hEGF. Result: The design and construction stages of pUC۵۷-۸xELP-Intein-hEGF cassette was successful and the accuracy of ۸xELP-Intein-hEGF was confirmed by sequencing. Conclusion: The use of ELP-intein cassette provides recombinant protein purification only with steps consisting of temperature, salt, and centrifugation, without need for proteolytic enzymes, and access to this technology provides the possibility of production and purification of recombinant proteins with minimum cost and facilities. 

Authors

Nogol Omoumi

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Mohammad-Ali Shokrgozar

National Cell Bank, Pasteur Institute of Iran, Tehran, Iran

Zahra Noormohammadi

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

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