Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein

• Published in: Protein expression and purification 2014-08, Vol.100, p.26-32
• Main Authors: Huang, Yu-Chieh, Chang, Hsun-Hui, Mou, Yun, Chi, Peter, Chan, Jerry Chun Chung, Luo, Shih-Chi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aragonite-associated protein; Base Sequence; Biomineralization; Calcite; Calcium Carbonate - metabolism; Chromatography, Affinity; Chromatography, Gel; Cloning, Molecular; Crystallization; Escherichia coli; Gastropoda - chemistry; Gastropoda - genetics; Gastropoda - metabolism; Genetic Vectors - genetics; Maltose-Binding Proteins - chemistry; Maltose-Binding Proteins - genetics; Maltose-Binding Proteins - isolation & purification; Maltose-Binding Proteins - metabolism; Molecular Sequence Data; Nacre - metabolism; Protein expression; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Vaterite
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2014.05.002

•The first report of soluble bacterially expressed aragonite-associated protein.•MALDI-TOF detects more than one Ca2+ binding sites present within AP7.•CD suggests the recombinant AP7 is partially unstructured at neutral pH.•CaCO3 crystallization assay suggest AP7 can inhibit the crystal growth of calcite. Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP–AP7 protein inE. coli (∼60mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP–AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.