Abstract
Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.
Highlights
Bile acids (BAs) are hydroxylated steroids synthesized by liver cells via a complex multistep process relying on the cytochrome P450-mediated oxidation of cholesterol [1,2].BAs are involved in important physiological functions as they facilitate the absorption of various intestinal nutrients and promote the biliary secretion of lipids, toxic metabolites, and xenobiotics [1,2]
According to the first reported protocol, chicken liver BABP (cL-BABP) was separated from the liver soluble proteins by performing two stages of gel filtration
The structural investigation of cL-BABP by NMR spectroscopy was reported using a recombinant protein expressed in E. coli [33]
Summary
Bile acids (BAs) are hydroxylated steroids synthesized by liver cells via a complex multistep process relying on the cytochrome P450-mediated oxidation of cholesterol [1,2].BAs are involved in important physiological functions as they facilitate the absorption of various intestinal nutrients (e.g., lipids and vitamins) and promote the biliary secretion of lipids, toxic metabolites, and xenobiotics [1,2]. The iLBP family is composed of various phylogenetically related, low molecular weight proteins including cellular retinol-binding proteins (CRBPs), retinoic acidbinding proteins (CRABPs), and fatty acid-binding proteins (FABPs) [7,8,9,10,11,12,13,14]. These proteins share a common structural architecture consisting of a ten-stranded antiparallel β-barrel and two α-helices enclosing the internal cavity where hydrophobic ligands bind [15,16,17,18,19,20,21]
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