Lecture 4 Protein Microarrays
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Lecture 4 Protein Microarrays

Course Number: PLSC 411, Fall 2009

College/University: Dickinson State

Word Count: 754

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Protein Expression Problems, Problems, Problems Issues in Protein Expression Two types of expression Homologous Heterologous 1 Homologous Expression Requires expression vector Transformation system Systems available Yeast Insect -- Sf9 cells Fungal -- Several species Mammalian Chinese Hamster Ovary cells HEK 293 Cells In many cases transformation is difficult Heterologous Expression Problems...

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Expression Problems, Protein Problems, Problems Issues in Protein Expression Two types of expression Homologous Heterologous 1 Homologous Expression Requires expression vector Transformation system Systems available Yeast Insect -- Sf9 cells Fungal -- Several species Mammalian Chinese Hamster Ovary cells HEK 293 Cells In many cases transformation is difficult Heterologous Expression Problems Toxic proteins Can use tight regulation of expression Missing modifications Addition of cofactors, metals, etc. Missing interactions Requires other proteins in complex to fold properly Missing tRNA Codon usage varies between organisms and kingodms 2 Heterologous Expression Problems Expressed proteins are improperly folded or mixed with other proteins Inclusion bodies Can be >= 90% of expressed protein. Protein is improperly folded and mixed with other proteins. Requires purification and refolding. Refolding is only partially successful http://web.mit.edu/king-lab/www/research/Scott/Scott-Research.html Protein Microarrays What do you want to know? 3 Types of arrays AM AM FM Sandwich ELISA FM Capture labeled haptens RP Purified recombinant proteins for P-P interactions or substrate --AM = Analytical micro array --FM = functional micro array --RP = Reverse Phase micro array Purified recombinant proteins to detect Abs Reverse phase M. Walther, B. Stillman, A. Frie and J Beator, Fast guide to protein microarrays, Whatman What is necessary to make an array? What is necessary to make an array? 1 or 2 specific antibodies for each protein All proteins to be studied are purified. A support and a detector What formats are available. Glass slide "chips" Nano-wells Nitrocellulose membranes 4 Antibody Production Inject purified protein into mice and produce monoclonal antibodies one at a time. Inject organism or mixture into mice and produce multiple monoclonal antibodies. In vitro antibody generation Phage display Expression library Expression Library Cells encoding Ig genes cDNA synthesis IgM and IgA producing cell lines cDNA Amplification of complementarity determining region (CDR) Pool of diverse DCRs Framework oligonucleotides E Sderlind, L Strandberg, P Jirholt, N. Kobayashi, V.Alexeiva, A-M berg, A Nilsson, Bo Jansson, M Ohlin, C Wingren, L Danielsson, R Carlsson, and C A.K. Borrebaeck 2000 Recombining germline-derived CDR sequences for creating diverse single framework antibody libraries Nature Biotechnology 18:852-856 5 Expression Library Framework oligonucleotides Pool of diverse DCRs Combination of oligonucleotides. Overlap extension gene synthesis. linker scFv antibody fragment gene Antibody limitations Specificity Binding strength Production 6 Genomic Protein Expression Major recombinational cloning Gateway strategies recombinational cloning system Gap repair-mediated recombination. Ligation independent cloning Genomic Cloning Strategies X Mix T4 pol +dC T4 pol +dG att B1 att B2 X att P1 Rxn 1 att P2 A B Mix att L1 att L2 C X att R1 Rxn 2 att R2 A - Gap repair-mediated recombination B - Ligation independent cloning C - Gateway recombinational cloning system E Phizicky, P. I. H. Bastiaens, H Zhu, M Snyder & S Fields 2003 Protein analysis on a proteomic scale Nature 422:208-215 7 Phage insertion/ recombination When the phage with att P site inserts into genome att B site it creates att L and att R sites. Excision of the phage results in the regeneration of the att B and att P sites. att P att B insertion att L att R excision att B att P Purification of Expressed Proteins Purification on a genomic scale requires an rapid and specific purification process. Typically used are Affinity tags Tandom Affinity Purification (TAP) Double tagging 8 Affinity Tags glutathione S-transferase Binds to glutathione coupled column Removed with glutatione maltose binding protein Binds to Maltose coupled column Removed with maltose hexyl histidine Binds to Ni+ chelating column Removed by addition of imidazole FLAG Binds to FLAG antibody column Removed by denaturation calmodulin binding protein Binds to calmodulin coupled column Strep II tag Binds to Avidin coupled column Can be removed with desthiobiotin protein A Binds to antibody column Removed by denaturation Expression systems Expression systems E. coli Insect cell line Sf9 Yeast (Pichia pastoris) Other cell lines. Issues RNA processing (alternative splici...
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