(1. School of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China; 2. Key Laboratory of Veterinary Biological Products Engineering, Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 3. School of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China; 4. Guotai Technology Innovation Center of Veterinary Biological Products (Taizhou), Taizhou 225300, China)
Abstract: Ferritin is a class of proteins ubiquitously found in various types of nucleated organisms and involved in the iron homeostasis regulation of body. Ferritin can self-assemble to form a hollow nanocage of about 14 nm in size and is often used as carrier for drug delivery and vaccine construction. The crystal structure of Mycoplasma ferritin shows atypical ferroxidase centres and new pathway for Fe2+ uptake, which are structurally different from those of other species. Not much work has been reported for Spiroplasma ferritin. Here, our studies show that Spiroplasma ferritin has low sequence similarity with other ferritins including Mycoplasma ferritins. Spiroplasma eriocheiris Ferritin (SeFer) was recombinant expressed in E. coli in soluble form after IPTG (isopropyl-beta-D-thiogalactopyranoside) induction. High-purity SeFer protein was obtained after purification by DEAE (diethylaminoethyl) weak anion exchange chromatography and gel filtration. Crystal screen of SeFer was performed by sitting drop vapor diffusion at 4℃ and 18℃. SeFer crystals grew in several screen conditions. The best crystal diffracted to 2.90 Å by synchrotron radiation source and it belonged to the I432 space group. The X-ray crystallography data collected here can be used for further structure determination. Native-PAGE, dynamic light scattering, and the position of peaks of SeFer in molecular sieve showed that the molecular weight of SeFer is about 400.0 kD, and the diameter of SeFer was about 15 nm, both of them are close to the theoretical value. The structural model of SeFer was predicted by using Alphafold 2. The predicted model shows that SeFer has a regular icosahedral overall structure, which is similar to other ferritins. However, SeFer model shows that the key residues of ferroxidase center differ from those of mycoplasma ferritin. Our results not only provide a basis for studying the crystal structure of SeFer, but also provide new candidates for vaccine vectors.
Key words: Spiroplasma eriocheiris; ferritin; protein purification; crystal structure
（Acta Laser Biology Sinica, 2024, 33(3): 227-235）