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ORIGINAL ARTICLE   

Minerva Biotecnologica 2017 September;29(3):126-32

DOI: 10.23736/S1120-4826.17.02225-X

Copyright © 2017 EDIZIONI MINERVA MEDICA

lingua: Inglese

Optimization of recombinant β-NGF purification using immobilized metal affinity chromatography

Zahra HAJIHASSAN 1 , Mehri ABDI 1, Elaheh ROSHANI YASAGHI 2, Azra RABBANI-CHADEGANI 3

1 Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran; 2 Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran; 3 Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran


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BACKGROUND: Several proteins have been purified in a single step from E. coli using fused histidine tails and immobilized metal affinity chromatography (IMAC). Many attempts have been done in order to optimize protein purification by this method.
METHODS: As the efficiency of purification is dependent on buffers and conditions used, so in this study different pH, imidazole concentration and incubation time were surveyed to optimize recombinant β-NGF purification.
RESULTS: In this work pET39b vector containing DsbA sequence was used in order to produce recombinant β-NGF with his tag tails; β-NGF is produced in the form of DsbA-βNGF fusion by using this vector. Also in order to determine the secondary structure and function of purified β-NGF, CD spectroscopy and treatment of PC12 cell line with β-NGF was done.
CONCLUSIONS: Our data represented the best strategies to increase the purification efficiency using IMAC. PC12 treatment with both purified recombinant β-NGF and DsbA-βNGF fusion indicated differentiation of this cell line to the nerve cells, demonstrating that DsbA-βNGF fusion retained β-NGF natural activity and is fully functional.


KEY WORDS: PC12 cells - Nerve growth factor - Isolation and purification

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