铁蛋白Ferritin原核表达和纯化及纳米颗粒胞外自组装

doi:10.11843/j.issn.0366-6964.2018.01.009

Abstract

Abstract:

This study aimed to express Helicobacter Pylori nonheme iron-containing Ferritin protein in Escherichia coli, and obtain the Ferritin nano-particles with natural nanostructure, for the application in disease and antigen presentation study by surface modification. To improve the expression of Ferritin, Ferritin gene was synthesized according to the Escherichia coli biased codon and cloned to the pET-32a vector. The recombinant protein was purified by affinity chromatography using His-trap Ni²⁺ column, and refold by gradient dialysis. The refolding protein was detected by transmission electron microscope to identify the nano-structure of the self-assembling protein. The results showed that the prokaryotic expression vector pET-32a-Ferritin was successfully constructed, and the recombinant His-Ferritin protein was expressed after induced by IPTG. The recombinant Ferritin protein was mainly expressed in the form of inclusion body. By use of His-trap Ni²⁺ affinity chromatography column, we obtained the recombinant Ferritin protein with 96% purity. The purified protein was in gradient dialysis to 2 mol·L^-1 urea buffer with a desalting column to obtain the refold protein. The refold protein had a nanostructure with the diameter of 12-20 nm under the transmission electron microscope, which was similar to the natural nanoparticles. In this study, the prokaryotic expression system of Ferritin was successfully established and the recombinant protein with nanostructure was prepared by induction expression, affinity chromatography purification and refolding. This study lays the foundation for the application of Ferritin in biomedical field.

CLC Number:

Q812

LI Zhi-peng, LIU Fu-hang, CUI Kui-qing, SHI De-shun, LIU Qing-you. Prokaryotic Expression and Purification of Ferritin and Nano-particles Self-assembling in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 75-82.

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Prokaryotic Expression and Purification of Ferritin and Nano-particles Self-assembling in vitro

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