Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.
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 7 kDa 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 (∼60 mg/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.
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
Yu-Chieh
YC
Huang
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
Hsun-Hui
HH
Chang
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
Yun
Y
Mou
Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan; Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan.
Peter
P
Chi
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
Jerry Chun Chung
JC
Chan
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan. Electronic address: scluo@ntu.edu.tw.
Shih-Chi
SC
Luo
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002119:Calcium Carbonate / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D049851:Gastropoda / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D060734:Nacre / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D058619:Maltose-Binding Proteins; D060734:Nacre; D011993:Recombinant Fusion Proteins; D002119:Calcium Carbonate
Aragonite-associated protein; Biomineralization; Calcite; Protein expression; Vaterite