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PROTEIN EXPRESSION IN MAMMALIAN CELLS ~ Techniques Workshop 23 May 2007

PROTEIN EXPRESSION IN MAMMALIAN CELLS ~ Techniques Workshop 23 May 2007. EXTRACELLULAR PROTEINS Large extracellular domains Modular multidomain organisation Posttranslational modifications Disulfide bridges Glycosylation. INTEGRIN α V β 3 9 domains 28 disulfide bridges

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PROTEIN EXPRESSION IN MAMMALIAN CELLS ~ Techniques Workshop 23 May 2007

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  1. PROTEIN EXPRESSION IN MAMMALIAN CELLS~Techniques Workshop 23 May 2007

  2. EXTRACELLULAR PROTEINS • Large extracellular domains • Modular multidomain organisation • Posttranslational modifications • Disulfide bridges • Glycosylation INTEGRIN αVβ3 • 9 domains • 28 disulfide bridges • 6 N-linked glycosylation sites

  3. VARIOUS EXPRESSION SYSTEMS • Cell free systems (RIKEN Genomic Sciences Center) • Prokaryotic • E. coli • Eukaryotic • Yeast cells • Insect cells • MAMMALIAN CELLS

  4. Nucleus RER ER Golgi PROTEIN EXPRESSION IN MAMMALIAN CELLS • S-S formation (ER) • Glycosylation (ER+Golgi) • Quality control (ER) ONLY CORRECTLY FOLDED PROTEINS ARE SECRETED

  5. COMMONLY USED MAMMALIAN CELLS HEK 293: Human embryonic kidney cells CHO: Chinese Hamster Ovary cells COS: Simian fibroblasts

  6. TISSUE CULTURE • Most mammalian cells are adherent • Cultured in plates or flasks • Grow in monolayer on specially treated surfaces • Medium supplemented with 5-10% Fetal Calf Serum • Laminar flow cabinet • CO2 incubator

  7. EXPRESSION VECTOR • Strong promoter (CMV) • Antibiotic resistance gene for selection of stable cell line • Antibiotic resistance gene for E. coli selection NOT ALWAYS INCLUDED BUT ESSENTIAL • Leader sequence

  8. TRANSFECTION OF MAMMALIAN CELLS • Electroporation • Ca-phosphate • Liposome based transfection reagents TYPES OF TRANSFECTION • Transient • Stable • Episomal

  9. TRANSIENT TRANSFECTION • Gene to protein in days • Testing expression • Functional studies • Low yield • Used in high-throughput structural studies (293 cells)

  10. STABLE TRANSFECTION • Gene to protein in ≥ 2 months • Complex process • Gene of interest integrates into genome of host cell • High yields (from 1 to 5 mg/l and higher) • Stock of cells expressing desired recombinant protein

  11. STABLE TRANSFECTION Transfection Selection pressure Screening clones for expression Cloning of positive clones Screening of single clones for expression Scaling up

  12. EPISOMAL TRANSFECTION • Gene to large scale protein production in ~ 4 weeks • Straightforward process • HEK EBNA cells (293 stably transfected with EBNA-1 gene) • EBNA-1 driven episomal replication ofOri-P containing vectors • Very high yields (5 to 20 mg/l and higher)

  13. EPISOMAL TRANSFECTION Transfection Selection pressure Scaling up (not clonal cell population)

  14. LARGE SCALE PROTEIN PRODUCTION Transfected cells grown to confluence in 10 x T175 flasks Wash with sterile PBS to remove contaminant proteins from serum (BSA) Culture cells in serum free medium (growth arrest) 3 x medium exchange every 48/76 hours CONDITIONED MEDIUM READY FOR PURIFICATION

  15. 500 mM Imidazole -45kDa 2500 2000 Absorption at 280 nm (mAU) 1500 1000 500 0 2000 1500 -45kDa Absorption at 280 nm (mAU) 1000 500 0 Vo 10 15 20 25 Elution volume (ml) EASY 2 STEPS PROTEIN PURIFICATION AFFINITY CHROMATOGRAPHY GEL FILTRATION

  16. GLYCOSYLATION • Mammalian sugar chains have highly complex structures • Good for functional studies • Big problem for protein crystallization SOLUTIONS • Mutagenesis of glycosylation sites • Enzymatic deglycosylation • Engineered cell lines (CHO Lec strains) • Chemical inhibitors of glycosylation pathway • Insect cells (simpler sugars)

  17. mut deg Lec wt wt -50kDa -40kDa -50kDa -50kDa -40kDa -40kDa DDR2 Receptor Tyrosine Kinase • 3 N-linked glycosylation sites in ectodomain • Predicted MW = 42 kDa Mutagenesis Enzymatic deglycosylation CHO Lec 3.2.8.1 Stable transfectant

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