Conference Program - ABRF 2011 - Association of Biomolecular ...

Poster Abstracts246 Purification of Bacterial APOA-1and Characterization of Novel Anti-Cancer Drug Delivery SystemT. Young 1 , A. Lacko 21North Carolina State University, Raleigh, NC,United States; 2 University of North Texas HealthScience Cente, Fort Worth, TX, United StatesAlthough chemotherapy regimens have proved effective in attackingcancer cells and tumors, side effects and drug resistance remain amajor concern during cancer therapy. The use of reconstituted highdensity lipoprotein (rHDL) nanoparticles has been investigated as adrug delivery system, including the transporting of small interferingribonucleic acid (siRNA). The use of rHDL nanoparticles has greatpotential, in this regard, due to their ability to specifically targetcancer cells via the HDL (SR-B1) receptor. The goal of these studieswas to improve the purification of Apolipoproteins A-1 (ApoA-1), amajor component of rHDL, and the preliminary characterization ofthe siRNA carrying rHDL nanoparticles. E.coli cells transfected withthe apo A-I gene was grown at 37OC, until an optical density of 0.6was reached. The cells were then induced with 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and centrifuged. Subsequently, thepellets were suspended in the lysate solution and loaded onto a Nickel-Sepharose column. Thereafter, rHDL nanoparticles using siRNA wereprepared. 160 mg per liter of purified ApoA1 was obtained. Particlemeasurements and the chemical composition of the particles are beinginvestigated. Further investigation regarding the efficiency of theincorporation of siRNA, the physical and chemical properties as wellas cytotoxicity of the particles will contribute to the assessment of theefficiency of these particles as a novel anti-cancer drug delivery system.247 Screening for Optimal PurificationConditions for Histidine-TaggedWater-Soluble and MembraneProteins Using Magnetic BeadsMethodsH. Hedlund, E. Mascher, G. RisbergGE Healthcare Life Sciences, Uppsala, SwedenThe development of methods for efficient expression screening ofmultiple numbers of recombinant proteins or optimization of thepurification conditions of proteins is essential to shorten the time fromgene to drug targets. The combination of para- magnetic beads andaffinity purification using IMAC resins (pre-charged with Ni2+-ions)for capture of histidine tagged proteins is an easy-to-use format anda powerful tool allowing for rapid, small-scale purification. We willdescribe two studies for optimization of purification conditions ofhistidine-tagged proteins. All experiments were done using Ni2+chargedHis Mag Sepharose Ni. In the first study optimal purificationconditions for a water-soluble histidine-tagged protein was investigatedat eight different buffer conditions and four different sample loads(varying from 25 % to 100 % of the total binding capacity). The studywas performed using a liquid handling workstation.A good balancebetween yield and purity was obtained at 40 mM imidazole and at asample load between 50 % and 100 % of the total binding capacity. Inthe second study screening for optimal solubilization and purificationconditions for an integral membrane protein was performed, usingseven detergents. The screening protocol involved a rapid purificationstep before analyses, evaluation and final choice of detergent(s).Obtained results from this screening gave reliable results that could alsobe fed into further scale-up experiments. We will present results fromWestern blotting, gel filtration and SDS-PAGE. The para-magnetic beadformat simplified and shortened the handling procedures through-outboth screening workflows.248 Production of Recombinant MouseFlower Protein in E. coli: Applicationof Mistic Fusion to Improving theExpression of Membrane ProteinsJ.L. Martinez-Torrecuadrada 1 , M. Marenchino 2 ,R. González 1 , J. López-Alonso 2 ,R. Campos-Olivas 2 , G. Roncador 3 , E. Moreno 41Proteomics Core Unit, Spanish National CancerCentre (CNIO), Madrid, Spain; 2 Spectroscopyand Nuclear Magnetic Resonance Unit. SpanishNational Cancer Centre (CNIO), Madrid, Spain;3Monoclonal Antibodies Core Unit, SpanishNational Cancer Centre (CNIO), Madrid, Spain;4Cell Competition Group, Spanish National CancerCentre (CNIO), Madrid, SpainThe structural and functional studies of membrane proteins havebeen greatly hindered due to difficulties in their over-expression andproduction. It is also often difficult to generate effective antibodies tomembrane proteins. In our laboratory, we have addressed the problemof producing high level amounts of membrane proteins in Escherichiacoli by use of Mistic, a Bacillus subtilis protein, as a fusion partner. Flower(Fwe) is a membrane protein that is conserved in animals and proposedto be a Ca2+ channel in neurons. It has been recently reported thatin Drosophila Flower is a component of the cell competition responsethat is required and sufficient to label cells as “winners” or “losers”,promoting the elimination of weaker cells from a growing populationin order to optimize tissue fitness. This process may have biomedicalimplications because imbalances in cell fitness appear during aging,cancer formation and metastasis. In this work, we employed themembrane protein Mistic to assist in the production of this protein.The construct 6xHis-Mistic – Flower carrying a TEV cleavage site wasefficiently overexpressed in E. coli. The IMAC-based purification andcleavage of the recombinant protein was achieved in the presence ofthe detergent lauryldimethylamino oxide (LDAO). Circular dichroismshowed a high content in helical structure as predicted from the aminoacid sequence by the program TMHMM. Also, the recombinant Mistic–Flower was used as antigen source to produce monoclonal antibodies inKO mice. The generated monoclonal antibodies were able to recognizeFlower protein by Western blot and immunohistochemistry, indicatingthat this recombinant protein retained the antigenicity of the nativeform. These antibodies will facilitate importantly further functionalstudies on Flower protein.102 • ABRF 2011 — Technologies to Enable Personalized Medicine