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Iranian Journal of Biotechnology، جلد ۱۶، شماره ۱، صفحات ۴۸-۵۸
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عنوان انگلیسی Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
چکیده انگلیسی مقاله Background: The increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.Objectives: Here, the expression pattern of α-Luffi n, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the eff ect of low temperature cultivation on the increase of α-Luffi n solubility was determined.Materials and Methods: The encoding cDNA for mature α-Luffi n was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purifi cation of recombinant protein was performed by Ni-NTA column.Results: In fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffi n was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.Conclusions: Our founding showed that EnBase had a greater effi cacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffi n without the need for refolding.
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نویسندگان مقاله Farzaneh Barkhordari |
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Mozhgan Raigani |
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Yeganeh Yeganeh Talebkhan Garoosi |
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Fereidoun Mahboudi |
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Fatemeh Davami |
Biotechnology Research center, Pasteur Institute of Iran, Tehran, Iran

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