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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Dickinson State - PLSC - 411
Protein-Protein InteractionsThe first question is why would we want to know about protein-protein interactions? This kind of information would allow us to determine if a protein is in a complex, a metabolic of signaling pathway, or involved with oth
Dickinson State - PLSC - 411
Protein-Protein interactionsWho are your buddies?Why are protein-protein interactions important? Identify proteins in complexes. Identify proteins that are in a metabolic or signaling pathway. Identify members of a non-enzymatic structure.1
Dickinson State - PLSC - 411
Functional Genomics Genome and EST sequencing can tell us how many POTENTIAL genes are present in the genome Proteomics can tell us about proteins and their interactions The goal of functional genomics is to define the function of each and every gene
Dickinson State - PLSC - 411
Sequence mutagenesis Advantages: Efficient and cost-effective method to generate a large mutant population Multiple allelic variants of the same gene can often be isolated Relatively random distribution of mutations in the genome Disadvantages: Time
Dickinson State - PLSC - 411
Microarrays and Transcript Profiling Gene expression patterns are traditionally studied using Northern blots (DNA-RNA hybridization assays). This approach involves separation of total or polyA+ RNA on agarose gels and after transfer to a nitrocellulo
Dickinson State - PLSC - 411
Terminologies Reporter: the nucleotide sequence present in a particular location on the array (a.k.a. probe) Feature: the location of a reporter on the array Composite sequence: a set of reporters used collectively to measure an expression of a pa
Dickinson State - PLSC - 411
Statistical analysis of microarray data Reference sample approaches Control (reference) vs. experimental competitive hybridization Ratio of intensities Twofold induction or repression significance Arbitrary threshold Microarrays do not provide data o
Dickinson State - PLSC - 411
Statistical analysis of microarray dataFigure 4. Pre-analysis data transformation using the spatial lowess function. The upper left panel shows log ratios of raw intensity data on a red-green color scale plotted on the spatial coordinates of the ar
Dickinson State - PLSC - 411
Serial Analysis of Gene Expression (SAGE) SAGE analysis is a method derived to provide a read-out, via sequencing, of the spectrum of genes being expressed in a cell. A method for comprehensive analysis of gene expression patterns Serial sequen
Dickinson State - PLSC - 411
REVIEWSTHE ART AND DESIGN OF GENETIC SCREENS: ARABIDOPSIS THALIANADamian R. Page and Ueli GrossniklausMolecular genetic studies rely on well-characterized organisms that can be easily manipulated. Arabidopsis thaliana - the model system of choice
Dickinson State - PLSC - 411
REVIEWSGENOME ANNOTATION: FROM SEQUENCE TO BIOLOGYLincoln SteinThe genome sequence of an organism is an information resource unlike any that biologists have previously had access to. But the value of the genome is only as good as its annotation. I
Dickinson State - PLSC - 411
Fischer-Vize, Science 270, 1828 (1995). 35. T. C. James and S. C. Elgin, Mol. Cell Biol. 6, 3862 (1986); R. Paro and D. S. Hogness, Proc. Natl. Acad. Sci. U.S.A. 88, 263 (1991); B. Tschiersch et al., EMBO J. 13, 3822 (1994); M. T. Madireddi et al., C
Dickinson State - PLSC - 411
Gene finding and Genome annotation What is a Gene? An inheritable trait associated with a region of DNA that codes for a polypeptide chain or specifies an RNA molecule which in turn have an influence on some characteristic phenotype of the organism.
Dickinson State - PLSC - 411
PERSPECTIVESGENOMICSDefining Genes in the Genomics EraMichael Snyder and Mark GersteinAgenome is defined as the entire collection of genes encoded by a particular organism. But what is a gene? Historically, the term gene, attributed to Johans
Dickinson State - PLSC - 411
REVIEWSGENOME ANNOTATION: FROM SEQUENCE TO BIOLOGYLincoln SteinThe genome sequence of an organism is an information resource unlike any that biologists have previously had access to. But the value of the genome is only as good as its annotation. I
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Idaho - ME - 433
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