Characterization of the oligomerization and ligand-binding properties of recombinant rat lipocalin 11 |
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Authors: | Yina Gu Qiang Liu Peiyan Chen Chenyun Guo Yan Liu Yufen Zhao Yonglian Zhang Donghai Lin |
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Institution: | 1. The key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China;2. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;3. Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China;4. Laboratory of Biomolecular NMR, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China |
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Abstract: | Lipocalin 11 (Lcn11), a recently identified member of the lipocalin family, potentially plays crucial physiological roles in male reproduction. In this present work, we cloned, expressed and purified the rat Lcn11 (rLcn11) protein in Escherichia coli. A C59A/C156A substitution was introduced to ameliorate the misfolding and aggregation problem associated with the wild-type protein. From circular dichroism and non-reducing SDS–PAGE, we characterized the conformational properties of rLcn11 as a typical lipocalin scaffold with the conserved disulfide bridge. The results obtained from size-exclusion chromatography, cross-linking experiment and dynamic light scattering analysis indicate that the recombinant rLcn11 protein forms dimer in neutral solution. By using fluorescent probe 8-anilino-1-naphtahlene sulfonic acid (ANS), we found rLcn11 might contain multiple hydrophobic binding sites for ligand binding. Similarly to the odorant-binding protein, rLcn11 processes a moderate affinity for binding 1-aminoanthracene (AMA), implying that Lcn11 might work as a dimeric chemoreception protein in male reproductive system. |
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Keywords: | rLcn11 rat lipocalin 11 L-PGDS lipocalin-type prostaglandin D synthase OBP odorant-binding protein MUP major urine protein PBP pheromone-binding proteins WT wild-type LB Luria-Bertani IPTG isopropyl β-d-thiogalactoside MALDI-TOF matrix-assisted laser desorption ionization time-of-flight DLS dynamic light scattering EGS Ethylene glycolbis (succinimidylsuccinate) CD circular dichroism |
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