Abstract: 【Objective】Investigate the effect of N-terminal elongation on the structural stability and protein self-assembly of the nano-cage bacterioferritin to provide the basis for the construction of functional nanomaterials by incorporating biomolecules at the N-terminus of bacterioferritin.【Method】Escherichia coli bacterioferritin protein was genetically engineered by elongating the N-terminus with six histidine residues. The mutant oligomerization state, secondary structure and thermo-stability were characterized and compared with that of the wild type using size exclusion chromatography, native gel electrophoresis and circular dichroism spectroscopy.【Result】The wild type formed a mixture of 24-mer and dimer in solution, while the mutant formed solely dimer. Consistent with the size exclusion chromatography experiment, only one band was observed corresponding to the wild-type dimer according to the native gel electrophoresis. Characterization of the mutant using circular dichroism demonstrated that it still folded into α-helical structure, exhibiting similar secondary structure as compared to the wild type. The melting temperature of the mutant was found to be 1.1 ℃ lower than that of the wild type. 【Conclusion】Although it still folded into the α-helical structure, the protein with an elongated N-terminus completely lost the ability to form the nano-cage structure, and formed dimer solely in solution with reduced thermo-stability compared with that of the parent protein. This study indicates that N-terminal elongation might result in a conformational change of bacterioferritin subunits, preventing the protein from self-assembling into the nano-cage structure.