Cultivation of KODO MILLET . made by Ghanshyam pptx
BIO Philadelphia yeast expression 2005
1.
2. Novel Solutions to Yeast Recombinant
Protein Expression
Dr Stephen Berezenko
Bio 2005 Philadelphia
June 21 2005
3. Issues with yeast expression
“S. cerevisiae glycosylation isn’t the same as higher
eukaryotes”
– True
– O-linked glycosylation
• Can be effectively controlled by pmt mutations and
downstream processing
– N-linked glycosylation
• Think smart - make the non-glycosylated protein
• In majority of examples still active
4. Misconceptions
• “Stable yeast episomal plasmids not available”
– Whole 2µm plasmids are very stable in selective
media
– Superior alternative to integration
• Curing and retransformation
• “S. cerevisiae has a limited secretion capacity”
– Significant inter-strain variation
– Strain engineering is not only possible, but highly
desirable
• Control proteolysis
• Increase expression
– Chemical mutagenesis & selection
– Endogenous gene over-expression
5. Enhanced Productivity
Protein Secreted Intracellular
Albumin 3 g/L WC *
Transferrin (N413Q, N611Q) 1 g/L WC *
scFv 3.6 g/L SN †
scFv-albumin 5.5 g/L SN †
Albumin-GSlinker-scFv 5.1 g/L SN †
Haemoglobin 2% CDW #
PAI-2 20% TSP ‡
Thymidine Phosphorylase 10% TSP ‡
α1-antitrypsin 40% TSP ‡
* WC: Whole culture
† SN: Supernatant
# CDW: Cell Dry Weight
‡ TSP: Total Soluble Protein
6. Expression System Performance
Delta Saccharomyces
cerevisiae expression
(g.L
-1
) Titre
(g.L
-1
)
P. pastoris 0.011
P. pastoris 0.049
S. cerevisiae ~0.0015
S. cerevisiae ~0.0015
S. cerevisiae 0.009
S. cerevisiae 1.3
Transferrin
(N413Q, N611Q)
Albumin 4.0-4.5 P. pastoris ~2.8
scFv-albumin fusion 5..5 P. pastoris ~0.010
~0.050
hGH 1.3
3.3 P. pastoris
Protein Competitive yeast systems
Yeast
7. Recombinant Human Albumin
• Large secreted
protein
– 67kDa
– 585 amino acids
• Highly folded
– 35 cysteines
– 17 disulphide bonds
– 1 free cysteine
Structure of rHA with five molecules of
myristate bound.
Curry et al. (1998) Nature Structural
Biology 5, 827-835
8. Yeast – Positive Attributes
• GRAS status
– S. cerevisiae
– K. lactis
• Wide range of strains
• Extensive industrial history
– 16 S. cerevisiae therapeutic
products marketed
– 7 P. pastoris therapeutic
products under
development
Gerngross, T. (2004) Nature
Biotechnology 22, 1409-1414
8m3
working volume fermentation vessel
Nottingham, U.K.
9. Scale-up and Technology Transfer
• Scale up
– R&D – 10L Fed-batch process
– Commercial – 12m3 (total volume)
– 8m3 (working volume)
– cGMP/FDA
• Technology Transfer
– Successfully completed to Japanese
Pharmaceutical company
– HGSI and albumin-based fusions
11. Albumin Fusions Proteins
• Albumin joined to another protein
through a peptide bond
–Sequence encoding a given therapeutic protein is
ligated to the sequence encoding human albumin
–High yield expression of the fusion protein (multiple
g/L) in optimised yeast strains
• Albumin has characteristics (charge
distribution and size of ~70kDa) that
prevent clearance via the kidney:19 day
half-life
12. What type of fusions can you make?
• The DNA sequence for the protein of
choice can be joined to the:
– C-terminus HSA
– N-terminus HSA
– In the middle
– Combinations
• So junction site of the fusion protein
can be defined at the molecular level
24. High Cell Density Fermentation System
• Synthetic chemical defined
– Simple, commercial grade materials
– No animal or human derived products
• Fed-batch process
• 5L batch
• 5L feed
• 300C ± 10C
• pH5.5 ± 0.1
• 1500rpm max
25. Expression time course
Analysis of culture supernatant
1 2 3 4 5 6
1ug
1ug
Lane
Feed Time
(hr)
Feed Vol
(L)
Biomass
(g CDW/L)
1 6.5 0.1 8.9
2 14.0 0.3 14.9
3 30.5 1.1 46.8
4 38.3 1.9 67.5
5 54.5 4.8 101.8
6 55.5 5.0 101.3
12% Bis-Tris SDS Novex gel
MES Buffered
27. Downstream Process Improvement
through Expression Strain Modifications
YAP3
yap3
rHA
monomer
45kDa
fragment
-Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro
•45kDa N-terminal fragment
•Observed in Pichia sp,
Kluyveromyces sp and Hansenula sp
•Carboxy terminus heterogeneous
•Terminating between Phe403 and Val409;
most common Leu407 and Val409
28. Downstream Process Improvement
through Expression Strain Modifications
• N-linked glycosylation
– None
• O-linked glycosylation
– Undetectable by ES-MS
– Approx. 0.7% of rHA bound to
ConA
– Average of 3-5 moles/mole
– Dolichyl-phosphate-D-mannose:
protein-O-D-mannosyltransferase
(PMT1 – 6)
• ConA binding material reduced
approx. five-fold in a pmt1
mutant yeast strain
α1-3
S/T
MNN1
PMT1-PMT6
MNT1/KRE2
α1-2
α1-3
α1-2
ER Lumen
29. Downstream Process Improvement
through Expression Strain Modifications
• Hsp150p (Pir2p)
– Host cell wall protein
– Large
• ~150kDa
• extensively O-linked
glycosylated
• 47kDa deglycosylated
– Removed by gel
permeation
chromatography
– Antigenic in yeast
sensitive subjects
Enrichment by ConA
chromatography
HSP150+ HSP150-
0.2mg
2mg
10mg
0.2mg
2mg
10mg
Western blot with anti-Hsp150p
30. Translational read-through
L G L stop A L D F F A R G 34aa S K stop
TTA GGC TTA TAA GCT TTG GAC TTC TTC GCC AGA GGT...........TCT AAA TAA ..
C-Terminus Albumin ADH1 Terminator
L G L stop stop A stop
TTA GGC TTA TAA TAA GCT TAA TCC ..........
C-Terminus Albumin ADH1 Terminator
Anti-Adh1p immunoaffinity purification
rHA-Adh1p rHA
Load
FlThru
Eluate
Load
FThru
Eluate
• Estimated translational read-through
– 0.002% (w/w) rHA-Adh1p fusion
32. Summary
• Whole 2µ episomal plasmid systems have high
mitotic stability
• Inter-strain variation
• Strain improvement is obtainable
– Increased productivity
– Control of post-translational modifications
– Improved downstream processing
• Chemically defined media
– No animal or human derived products
– Robust and reproducible high cell density fermentation
• Simplicity
– Significantly improves scale-up and technology transfer
