Expression of biologically active rat apolipoprotein AIV in Escherichia coli Min Liu a, * , Nick Maiorano a , Ling Shen a , Kevin Pearson a , Daisuke Tajima a , Dian Ming Zhang a , Stephen C. Woods b , Randy J. Seeley b , W. Sean Davidson a , Patrick Tso a a Department of Pathology and Laboratory Medicine, University of Cincinnati School of Medicine, Cincinnati, OH 45267-0529, USA b Department of Psychiatry, University of Cincinnati School of Medicine, Cincinnati, OH 45267-0529, USA Received 6 May 2002; received in revised form 31 July 2002; accepted 15 October 2002 Abstract 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 AIVelicited 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. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Apolipoprotein A-IV; Recombinant expression; Escherichia coli; Food intake 1. Introduction Apolipoprotein AIV (apo AIV) is a major component of chylomicrons and high-density lipoproteins. Rat apo AIV is synthesized in the small intestine and liver, whereas human apo AIV is predominantly found in the small intestine [9,22]. Rat apo AIV protein, a 43-kDa protein, has an 1173-nucleotide coding region that specifies a protein of 391 amino acids. The portion of the cDNA sequence representing the mature plasma protein contains a 66-nuc- leotide subsequence that is repeated at least 13 times. Although each repeated unit has some sequence variation, base changes in comparably positioned codons generally conserve the chemical type, if not the identity, of the corresponding amino acids [1]. Several studies have provided evidence that apo AIV modulates plasma cholesterol and lipoprotein metabolism [4,8], upper gastrointestinal function [15,16], control of food intake [5–7], protection against lipoprotein oxidation [19] and the risk for atherosclerosis [3,17]. Apo AIV mRNA and protein were recently identified in the hypothalamus of rats, and mRNA levels were found to be regulated physiologic- ally [13]. Previous experiments in which apo AIV was adminis- tered relied upon extracted and purified apo AIV from rat plasma [10]. However, native rat apo AIV is present in relatively low levels in both plasma and lymph, and the process of purifying it is both tedious and labor intensive, rendering large-scale preparation of this apolipoprotein difficult. Therefore, accomplishing high-level expression of the protein using recombinant DNA technology is a desirable alternative. The purpose of the current study was to develop a reliable and efficient expression system for generating recombinant rat apo AIV in Escherichia coli. The biological activity of the recombinant apo AIV protein generated was determined in an acute feeding assay after intracerebroventricular injection. We further demonstrated that neither native rat nor recombinant rat apo AIV elicit a 0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII:S0031-9384(02)00959-9 * Corresponding author. Tel.: +1-513-558-4957; fax: +1-513-558- 2141. E-mail address: lium@email.uc.edu (M. Liu). Physiology & Behavior 78 (2003) 149 – 155