TY - JOUR
T1 - Comparative ligand-binding analysis of ten human lipocalins
AU - Breustedt, Daniel A.
AU - Schönfeld, Dorian L.
AU - Skerra, Arne
N1 - Funding Information:
The authors wish to thank Amber Nasreen, Nicole Bischoff, and Martin Distel for providing lipocalin expression vectors and Anjali Karande (Indian Institute of Science, Bangalore, India) for the cloned GLY cDNA. E. coli strain MC4100Δskp was kindly provided by Matthias Müller (Universität Freiburg, Germany). The preparation of mutant streptavidin Sepharose by Ina Theobald and Irmgard Neumaier is gratefully acknowledged. This work was supported by PIERIS Proteolab AG, Freising-Weihenstephan, Germany.
PY - 2006/2
Y1 - 2006/2
N2 - At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), α1-acid glycoprotein, α1- microglobulin, apolipoprotein D, β-trace protein, complement component 8γ, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and β-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.
AB - At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), α1-acid glycoprotein, α1- microglobulin, apolipoprotein D, β-trace protein, complement component 8γ, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and β-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.
KW - ANS
KW - DAUDA
KW - E. coli secretion
KW - Fluorescent probe
KW - Retinoic acid
KW - Retinol
KW - Retinol-binding protein
KW - Strep-tag
UR - http://www.scopus.com/inward/record.url?scp=33644506112&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2005.12.006
DO - 10.1016/j.bbapap.2005.12.006
M3 - Article
C2 - 16461020
AN - SCOPUS:33644506112
SN - 1570-9639
VL - 1764
SP - 161
EP - 173
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 2
ER -