Expression of biologically active rat apolipoprotein AIV in Escherichia coli.
Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.
Liu
Min
M
Department of Pathology, University of Cincinnati School of Medicine, Cincinnati, OH 45267-0529, USA. lium@email.uc.edu
Maiorano
Nick
N
Shen
Ling
L
Pearson
Kevin
K
Tajima
Daisuke
D
Zhang
Dian Ming
DM
Woods
Stephen C
SC
Seeley
Randy J
RJ
Davidson
W Sean
WS
Tso
Patrick
P
D000595:Amino Acid Sequence; D000818:Animals; D001054:Apolipoproteins A / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D001362:Avoidance Learning / Q000187:drug effects; D001835:Body Weight / Q000187:drug effects; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004435:Eating / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D015151:Immunoblotting; D007276:Injections, Intraventricular; D008074:Lipoproteins / Q000737:chemistry / Q000302:isolation & purification; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013649:Taste / Q000187:drug effects
D001054:Apolipoproteins A; D018076:DNA, Complementary; D008074:Lipoproteins; D011994:Recombinant Proteins; C022205:apolipoprotein A-IV