Expression and purification of recombinated ferritin nanocages文献综述

BACKGROUND

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.

It is not possible to characterize the interactions or functions of a protein without protein purification. This is because the protein could contain binding proteins or other components that alter its state. If you skip protein purification, then there is no way to accurately gauge the quality of the protein.

Apoferritin is a protein commonly present in the intestinal mucosa membrane. The important biological function of apoferritin is its ability to bind and store iron, by combining with a ferric hydroxide–phosphate compound to form ferritin. Apoferritin from horse spleen was the first protein cage studied and used as a template for the synthesis of inorganic nanoparticles. Apoferritin has found extensive use in the synthesis of numerous metallic nanoparticles including Pd² , Cu² , Fe² , Co² , Cr³ , Ni² , Mn² , Cd² , and Zn² . Aime et al. (2002) reported that gadolinium-loaded apoferritin was used as a contrast agent for MRI. Sanchez et al. (2009) reported that water-soluble apoferritin-encapsulated gadolinium nanoparticles were used as a contrast agent for MRI. Kaacute;lmaacute;n et al. (2010) reported that manganese (Mn)-loaded apoferritin was also used as a contrast agent for MRI. Makino et al. (2011) also reported that apoferritin-encapsulated gadolinium (Gd) was used as a contrast agent for MRI. Geninatti Crich et al. (2012) reported that manganese (Mn)-loaded apoferritin was used as an MRI probe for the detection of hepatocarcinoma lesions. Bradshaw et al. (2013) reported that an apoferritin-encapsulated lead sulfide (PbS) quantum dots nanocomposite was used as a non-invasive probe for imaging colorectal carcinoma (CRC) cells. Recently, Valero et al. (2014) reported that that a maghemite nanoparticle coated with apoferritin protein capsid was used as a contrast agent for MRI.

MATERIALS

Plasmid pET-30a ( )/ FTH was synthesized by GenScript Biotechnology Co., Ltd (Nanjing, China). Host strains of
Escherichia coli ArcticExpresstrade; (DE3) were produced in our laboratory. Kanamycin and isopropyl beta;-D-1-thiogalactopvranoside (IPTG) were purchased from Sangon Biotech Co., Ltd (Shanghai, China). His·Bind Purification Kit was obtained from Merck Millipore (Merck, Germany).

Methodology

Luria Bertani (LB) Medium Preparation

At first, weigh out 2g Tryptone, 1g Yeast extract and 2g NaCl. Mixed them properly and then add into a 500 mL erlenmeyer flask. Measure out approximately 200mL of distilled water into the erlenmeyer. After measuring it, shake the bottle to dissolve the reagents. Once the reagents have fully dissolved, adjust the pH. Then sterilization, autoclave the solution on a liquid cycle (121 degrees C for 20min).

Protein expression steps: