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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 • 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 Capture labeled haptens Sandwich ELISA FM FM AM 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. Friße 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 Söderlind, L Strandberg, P Jirholt, N. Kobayashi, V.Alexeiva, A-M Åberg, 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 strategies – Gateway recombinational cloning system – Gap repair-mediated recombination. – Ligation independent cloning Genomic Cloning Strategies att B1 X X Mix T4 pol +dC att B2 Rxn 1 att P1 att P2 att L1 att L2 T4 pol +dG A B Mix 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 λ Phage insertion/ recombination • • When the λ 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 – hexyl histidine • Binds to Ni+ chelating column • Removed by addition of imidazole – calmodulin binding protein • Binds to calmodulin coupled column – protein A • Binds to antibody column • Removed by denaturation – maltose binding protein • Binds to Maltose coupled column • Removed with maltose – FLAG • Binds to FLAG antibody column • Removed by denaturation – Strep II tag • Binds to Avidin coupled column • Can be removed with desthiobiotin Expression systems • Expression systems – E. coli – Insect cell line Sf9 – Yeast (Pichia pastoris) – Other cell lines. • Issues – RNA processing (alternative splicing) – Protein processing (modifications) • Phosphorylation, cofactors, etc. – Solubility – Toxicity 9 Attachment of proteins • Want proteins to – Maintain structure and functionality • Includes keeping access to functional site – Achieve maximal binding density • For some applications nanowells are needed. Attachment of proteins • Different surfaces – Random attachments • Covalent: amines, aldehyde- and epoxyderivatized glass surfaces • Non-covalent: nitrocellulose, gel pads, or poly-Llysine • Affinity tagged surfaces (oriented) – Nickel – Anti tag antibodies 10 Detection • small molecule probes – fluorescent, affinity, photochemical, or radioisotope tags – Uses common DNA array devices for detection • label-free detection – Surface plasmon resonance (SPR) • Detects changes in index of refraction • Sensitivity 1 nM (~100 fold less than fluorescence) – carbon nanotubes and nanowires (conductance) – Micro-electromechanical systems cantilevers Protein Microarray Uses • Interactions/reactions – Protein-protein – Protein-DNA/RNA – Protein-lipid – Protein-drug • Disease protein markers • Protein substrate detection – Phosphorylation 11