| It is generally accepted that the plasma membrane of eukaryotic cells is organized into microdomains. Lipid rafts are newly discovered liquid-ordered microdomain enriched in sphingolipids and cholesterol. In lipid rafts, there are many proteins involved in signal transduction. Additionally, lipid rafts have some specific structural proteins, including flotillin (reggie), caveolin and stomatin. Lipid rafts play an important role in a wide range of biological processes, including numerous signal transduction pathways, apoptosis, adhesion, migration, synaptic transmission, organization of the cytoskeleton, and protein sorting during both exocytosis and endocytosis.Flotillins (reggies), the major specific structural proteins of biomembrane microdomain lipid rafts, consist of two members:flotillin-1 (reggie-2) and flotillin-2 (reggie-1). Similar to stomatin, prohibitin, and bacterial membrane proteins HfLK and HfLC, flotillins consist of conserved SPFH domain in the N-terminus, which mediates the cellular processes and regulates the proteins at lipid rafts, including linkage of lipid rafts to the actin cytoskeleton. In addition, the flotillins contain another conserved flotillin domain in the C-terminus, which is not present in other SPFH domain-containing proteins. The flotillin domain in the C-terminal is predicted to form coiled-coil structures. Via this domain, the flotillins form homo-and hetero-oligomers.Current researches of flotillin-2 mostly focused on its function in signaling processes by increasing or knocking down the flotillin-2's expression level, but the regulation of flotillin-2 was seldom investigated. In order to identify flotillin-2's regulator or effecter, we performed yeast two-hybrid screens to identify novel interact proteins of flotillin-2. One candidate isolated is ankyrin repeat and SOCS box containing protein 7 (ASB-7), a member of the ankyrin repeat and SOCS box containing protein family. The SOCS box mediates interactions with an Elongin B/ Elongin C/cullin 5/Roc protein complex to constitute a functional E3 ubiquitin ligase complex, while the N-terminal ankyrin repeat region recruits substrate proteins to mediate substrate polyubiquitination and proteasome-mediated degradation.The interaction was further verified by in vivo co-localization and in vitro pull down binding assays. A series of vectors encoding flotillin-2 and ASB-7's different domains were constructed to performed co-localization and pull down binding assays. The interaction site of flotillin-2 and ASB-7 was mapped to flotillin-2's SPFH domain and ASB-7's ankyrin repeat domain. Cotransfection of ASB-7 and flotillin-2 showed that the increased expression of ASB-7 reduced the expression level of flotillin-2. Thus ASB-7 may down regulate flotillin-2's expression level by targeting it for polyubiquination.Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, it is difficult to express flotillin-2 as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags:poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included 2 steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1.
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