protein expression in mammalian cells techniques workshop 23 may 2007 l.
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PROTEIN EXPRESSION IN MAMMALIAN CELLS ~ Techniques Workshop 23 May 2007 PowerPoint Presentation
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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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slide2
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
slide3

VARIOUS EXPRESSION SYSTEMS

    • Cell free systems (RIKEN Genomic Sciences Center)
    • Prokaryotic
      • E. coli
    • Eukaryotic
      • Yeast cells
      • Insect cells
      • MAMMALIAN CELLS
slide4

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

slide5
COMMONLY USED MAMMALIAN CELLS

HEK 293: Human embryonic kidney cells

CHO: Chinese Hamster Ovary cells

COS: Simian fibroblasts

slide6
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
slide7
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
slide8
TRANSFECTION OF MAMMALIAN CELLS
  • Electroporation
  • Ca-phosphate
  • Liposome based transfection reagents

TYPES OF TRANSFECTION

  • Transient
  • Stable
  • Episomal
slide9
TRANSIENT TRANSFECTION
  • Gene to protein in days
  • Testing expression
  • Functional studies
  • Low yield
  • Used in high-throughput structural studies (293 cells)
slide10
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
slide11

STABLE TRANSFECTION

Transfection

Selection pressure

Screening clones for expression

Cloning of positive clones

Screening of single clones for expression

Scaling up

slide12
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)
slide13

EPISOMAL TRANSFECTION

Transfection

Selection pressure

Scaling up

(not clonal cell population)

slide14

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

slide15

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

slide16
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)
slide17

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