Protein Expression in Mammalian cells

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

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
Selection pressure
Screening clones for expression
Cloning of positive clones
Screening of single clones for
expression
Transfection
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 of
Ori-P containing vectors
– Very high yields (5 to 20 mg/l and
higher)

13. EPISOMAL TRANSFECTION
Selection pressure
Scaling up
Transfection
(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. EASY 2 STEPS PROTEIN 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 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. 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
-50kDa
-40kDa -40kDa
-50kDa
-50kDa
-40kDa
wt wt
mut deg Lec