1. PEGS, 7th April 2009 (c) Marco Casteleijn Parallel high throughput expression of Thermostable Phosphorylases Focus on technology

2. 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 Helsinki Turku Tampere Lappeenranta Joensuu Kuopio Jyväskylä

3. PEGS, 7th April 2009 (c) Marco Casteleijn Biocatalysts - trends Davenport, R. VOL. 4 NO. 1 March 2008 INDUSTRIAL BIOTECHNOLOGY

4. 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)

5. 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”

6. PEGS, 7th April 2009 (c) Marco Casteleijn Biocatalysis at our Facilities Enzymes... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile 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 scale cultivations, on-line evaluation of proper folding Starting points Novel thermostable phosphorylases Development of High Throughput methods 45 gene cultivation product High Throughput parallel optimization

7. PEGS, 7th April 2009 (c) Marco Casteleijn Technology EnBase TM 1 [Johanna Panula-Perälä et al. 2008] (University of Oulu, BPEL) 96 well plates

8. PEGS, 7th April 2009 (c) Marco Casteleijn Technology Cytoplasmic expression library 2 [Kraft et al. 2007] Recombinant protein expression Expression vector library for Cytoplasmic Protein Expression & Optimization from Uwe Horn 2 45 different expression vectors 3 promoters 3 Ribosomal binding sites (SD) 5 different fusion tags PromotorRibosome-biding Site

9. PEGS, 7th April 2009 (c) Marco Casteleijn Generation of expression plasmids Transformation Protein expression Evaluation HT-cloning Online monitoring of protein aggregation 1 LucA reporter plasmid Gateway System Robotic Systems High cell density Cultivation Enbase Sampling, OD determination Transformations: ~ 500/day Protein expression: 60 x 4 x 2 (T 1 &T 2 ) = 480/experiment Online: OD 490 / Aggregation/ Sampling Technology High Throughput strategies

10. 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

11. PEGS, 7th April 2009 (c) Marco Casteleijn Thermostable Phosphorylases Simple and cheap Purification Higher general Stability Strategies for improved Protein stability Suitability for specific industrial Processes Structure- stability Relationships Evolutionary Significance of Thermophilic MO Basic research Industrial application Case study 1: The Phosphorylase 1 family

12. PEGS, 7th April 2009 (c) Marco Casteleijn Case study 1: The Phosphorylase 1 family Protein expression Activity assay by NMR(E. Coli UP in Cell Lysate)

13. 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 EnBase TM 24 DWP Outcome: Average final cell density over OD 600 =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 EnBase TM 24DWP … Case study 2: EnBase TM 24-DWP replaces traditional Shake Flasks

14. 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 ---------- high NH 4 + glucose 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: EnBase TM Flo

15. PEGS, 7th April 2009 (c) Marco Casteleijn Biocatalysis at our Facilities The 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  ongoing EnBaseTM Flo 46

16. PEGS, 7th April 2009 (c) Marco Casteleijn Acknowledgements Bioprocess Engineering Laboratory Prof. Peter Neubauer Ph.D Mari Ylianttila Kathleen Szekér Johanna Panula-Perälä Chemistry Department Ph.D. Sampo Mattila Silja Pelttari Nanna Alho Biochemistry Department Prof. Rik Wierenga Ph.D. Andre Juffer Niko Pursiainen Oulu University Academic Partners Technical University Berlin (GE) Prof. Peter Neubauer University of Kuopio (FI) Prof. Seppo Lapinjoki Prof. Igor Mikhailopulo Jarkko Roivainen Leibniz-Institute for Natural Product Research and Infection Biology – HKI (GE) Dr. Uwe Hörn Mario Kraft Industry Partners Biosilta (FI) High Throuput expression system Metkinen (FI) Modified Nucleosides Fermentas (LV) Methodology, Scale-up: Juozas Šiurkus