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PEGS, 7th April 2009(c) Marco Casteleijn
Parallel high throughput expression of Thermostable Phosphorylases
Focus on technology
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalyst platform
Faculty of Science:
Chemistry (3 groups)Biochemistry (1 group)
Faculty of Technology:
Bioprocess Engineering (1 group)
Linnanmaa campus
University of Oulu
OULU
Rovaniemi
Vaasa
HelsinkiTurku
Tampere
Lappeenranta
Joensuu
Kuopio
Jyväskylä
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalysts - trends
Davenport, R. VOL. 4 NO. 1 March 2008 INDUSTRIAL BIOTECHNOLOGY
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalysts - Bottlenecks
Metagenomics”finding Enzymes”
Protein Engineering”making enzymes”
Bioprocess Development”using enzymes”
• Finding the right markers (M)
• Vast amount of genetic data (M, PE) amount of DNA, processing many clones and sequences, library strategies
• Vast amount of gene products (M, PE, BD) purity, activity, selectivity
• Effective screening of activity (M, PE, BD)data mining, gene isolation, product isolation
• Adjustable product-gene expression, inteference-free operation (BD)
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalysts - Solutions
• Miniaturization
• Parallelization
• High Throughput approaches
• Modelling
• Bioinformatics
Metagenomics”finding Enzymes”
Protein Engineering”making enzymes”
Bioprocess Development”using enzymes”
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalysis at our FacilitiesEnzymes...
Biocatalysts
Thermostabilityexample moleculesphosphorylases
TIM barrelsversatile platform for isomerisation
BIOCAT-HT: Production of active thermostable phosphorylases based on High Throughput strategies
Parallel transformations and expressions of phosphorylases isolated from thermophilic organisms by using a fusion-partner plasmid library.
• High quantity approach: automated, fed-batch small scalecultivations, on-line evaluation of proper folding
• Starting points
•Novel thermostable phosphorylases
•Development of High Throughput methods
45
gene cultivation product High Throughput parallel
optimization
PEGS, 7th April 2009(c) Marco Casteleijn
TechnologyEnBaseTM
1 [Johanna Panula-Perälä et al. 2008] (University of
Oulu, BPEL)
96 well plates
PEGS, 7th April 2009(c) Marco Casteleijn
TechnologyCytoplasmic expression library
2 [Kraft et al. 2007]
Recombinant protein expression
Expression vector library for Cytoplasmic Protein
Expression & Optimization from Uwe Horn2
45 different expression vectors
• 3 promoters
• 3 Ribosomal binding sites (SD)
• 5 different fusion tags
PromotorRibosome-biding Site
PEGS, 7th April 2009(c) Marco Casteleijn
Generation of expression plasmids
Transformation
Protein expression
Evaluation
HT-cloning
Online monitoring ofprotein aggregation1
LucA reporter plasmid
Gateway System
Robotic Systems
High cell density CultivationEnbase
Sampling, OD determination
Transformations: ~ 500/dayProtein expression: 60 x 4 x 2 (T1&T2) = 480/experimentOnline: OD490 / Aggregation/ Sampling
TechnologyHigh Throughput strategies
PEGS, 7th April 2009(c) Marco Casteleijn
Case study 1: The Phosphorylase 1 family
Deinococcus geothermalis- Gram (+) Bacterium- Thermophilic radiophile- Optimal growth at 47°C - Isolated in hot springs in Italy and Portugal [Ferreira et al., 1997]
APE 2105.1 - UP
APE 0993.1 - MTAP
Dgeo 1497 - PNP
Aeropyrum pernix-Aerobic Archaeon- Optimal growth: 95°C- Isolated from hydrothermal vents in Japan [Sako at al., 1996]
- Genome: 67% GC content sequenced in 1999 [Kawarabayasi et.al, 1999]
reannoted in 2006 [Yamazaki et al., 2006]
*
* Poster:
Parallel high throughput expression
of Thermostable Phosphorylases
PEGS, 7th April 2009(c) Marco Casteleijn
Thermostable Phosphorylases
Simple and cheap Purification
Higher general Stability
Suitability for specific industrial
Processes
Strategies for improved Protein stability
Structure-stability Relationships
EvolutionarySignificance of
Thermophilic MO
Basic research Industrial application
Case study 1: The Phosphorylase 1 family
PEGS, 7th April 2009(c) Marco Casteleijn
Case study 1: The Phosphorylase 1 family
Protein
expression
Activity assayby NMR(E. Coli UP in Cell
Lysate)
PEGS, 7th April 2009(c) Marco Casteleijn
Partners: J.Ottosson, H. Tegel, M. Hjelmare; M. Uhlen KTH Stockholm (Human Protein Atlas)
Test of 15 random clones…..
Challenge: Saving time and manpower by scaling down the expression format of 4 x 72 SF (288 SF) per week to EnBaseTM 24 DWP
Outcome: • Average final cell density over OD600=25• Protein yield was best with MSM + booster (blue lines), cells are still in exponential phase• Preliminary results look promising that 6 shakes flasks can be replaced by one
EnBaseTM24DWP
05
10152025303540
0 10 20 30
OD
600
time [h]
strain 1
1
2
0
5
10
15
20
25
30
0 10 20 30
OD
600
time [h]
strain 5
1
2
0
5
10
15
20
25
30
0 10 20 30
OD
600
time [h]
strain 2
1
2
05
1015202530
0 10 20 30
OD6
00
time [h]
strain 3
1
2
0
5
10
15
20
25
30
35
0 10 20 30
OD
600
time [h]
strain 4
1
2
0
5
10
15
20
25
30
35
0 10 20 30
OD
600
time [h]
strain 6
1
2
0
5
10
15
20
25
30
0 10 20 30
OD
600
time [h]
strain 7
1
2
0
5
10
15
20
25
30
0 10 20 30O
D60
0
time [h]
strain 8
1
2
…
Ladder 1 2 3 4 5 6 7 8
Targetprotein
Case study 2: EnBaseTM 24-DWP replaces traditional Shake Flasks
PEGS, 7th April 2009(c) Marco Casteleijn Example: E. coli B21(DE3) and human PDI, 30°C, 180 rpm, EnBase
mini shake flask system
Challenge: High cell density cultivation on complex medium
Low ---------- pH ---------- highNH4
+glucose0
5
10
15
20
25
30
35
40
0 5 10 15 20 25
OD
600
time [h]
5
6
7
8
9
0 5 10 15 20 25
pH
time [h]
EnBase CM
EnBase MSM + booster
EnBase MSM +glucose +booster
Solution: Controlled growth on EnBase with MSM and boosting at time of induction
Case study 2: New developments: EnBaseTM Flo
PEGS, 7th April 2009(c) Marco Casteleijn
Biocatalysis at our FacilitiesThe right Tools for the Right Methods...
Tools
High Throughput* Hamilton pipetting station
Parallelization* Small scale cultivation technology (EnBase)* Parallel cloning library
Miniaturization * Cultivations* Parallel cloning library
New Methods
High Throughput transformation
High Throughput optimization of protein expression
From Small Scale to Large Scale without further optimization
High Throughput production of crystals for Crystallography ongoingEnBaseTM Flo
46
PEGS, 7th April 2009(c) Marco Casteleijn
Acknowledgements
Bioprocess Engineering Laboratory
Prof. Peter NeubauerPh.D Mari Ylianttila
Kathleen SzekérJohanna Panula-Perälä
Chemistry Department
Ph.D. Sampo Mattila
Silja PelttariNanna Alho
Biochemistry Department
Prof. Rik Wierenga Ph.D. Andre JufferNiko Pursiainen
Oulu
University
Academic
PartnersTechnical University Berlin (GE)
Prof. Peter Neubauer
University of Kuopio (FI)
Prof. Seppo LapinjokiProf. Igor Mikhailopulo
Jarkko Roivainen
Leibniz-Institute for Natural Product Research and Infection Biology – HKI (GE)
Dr. Uwe HörnMario Kraft
Industry
PartnersBiosilta (FI)
High Throuput expression system
Metkinen (FI)
Modified Nucleosides
Fermentas (LV)
Methodology, Scale-up: Juozas Šiurkus
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