15703696.PPT

15703696.PPT

• 1.
  PROTEIN EXPRESSION INMAMMALIAN CELLS ~ Techniques Workshop 23 May 2007
• 2.
  EXTRACELLULAR PROTEINS – Largeextracellular 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 –Cellfree systems (RIKEN Genomic Sciences Center) –Prokaryotic •E. coli –Eukaryotic •Yeast cells •Insect cells •MAMMALIAN CELLS
• 4.
  Nucleus ER RER Golgi ONLY CORRECTLY FOLDEDPROTEINS ARE SECRETED PROTEIN EXPRESSION IN MAMMALIAN CELLS – S-S formation (ER) – Glycosylation (ER+Golgi) – Quality control (ER)
• 5.
  COMMONLY USED MAMMALIANCELLS HEK 293: Human embryonic kidney cells CHO: Chinese Hamster Ovary cells COS: Simian fibroblasts
• 6.
  TISSUE CULTURE – Mostmammalian 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 – Strongpromoter (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 MAMMALIANCELLS – Electroporation – Ca-phosphate – Liposome based transfection reagents TYPES OF TRANSFECTION – Transient – Stable – Episomal
• 9.
  TRANSIENT TRANSFECTION – Geneto protein in days – Testing expression – Functional studies – Low yield – Used in high-throughput structural studies (293 cells)
• 10.
  STABLE TRANSFECTION – Geneto 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 Selection pressure Screeningclones for expression Cloning of positive clones Screening of single clones for expression Transfection Scaling up
• 12.
  EPISOMAL TRANSFECTION – Geneto large scale protein production in ~ 4 weeks – Straightforward process – HEK EBNA cells (293 stably transfected with EBNA-1 gene) – EBNA-1 driven episomal replication of Ori-P containing vectors – Very high yields (5 to 20 mg/l and higher)
• 13.
  EPISOMAL TRANSFECTION Selection pressure Scalingup Transfection (not clonal cell population)
• 14.
  LARGE SCALE PROTEINPRODUCTION 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.
  EASY 2 STEPSPROTEIN PURIFICATION AFFINITY CHROMATOGRAPHY GEL FILTRATION 0 500 Absorption at 280 nm (mAU) 1000 1500 2000 2500 500 mM Imidazole -45kDa Elution volume (ml) Vo 10 15 20 25 0 500 1000 1500 Absorption at 280 nm (mAU) 2000 -45kDa
• 16.
  GLYCOSYLATION – Mammalian sugarchains 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.
  DDR2 Receptor TyrosineKinase – 3 N-linked glycosylation sites in ectodomain – Predicted MW = 42 kDa Mutagenesis Enzymatic deglycosylation CHO Lec 3.2.8.1 Stable transfectant -50kDa -40kDa -40kDa -50kDa -50kDa -40kDa wt wt mut deg Lec