Parallel high throughput expression of Thermostable Phosphorylases Focus on technology

PEGS, 7th April 2009(c) Marco Casteleijn

Parallel high throughput expression of Thermostable Phosphorylases

Focus on technology


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


Biocatalysts - trends

Davenport, R. VOL. 4 NO. 1 March 2008 INDUSTRIAL BIOTECHNOLOGY


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)


Biocatalysts - Solutions

• Miniaturization

• Parallelization

• High Throughput approaches

• Modelling

• Bioinformatics

Metagenomics”finding Enzymes”

Protein Engineering”making enzymes”

Bioprocess Development”using enzymes”


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


TechnologyEnBaseTM

1 [Johanna Panula-Perälä et al. 2008] (University of

Oulu, BPEL)

96 well plates


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


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


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


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




Protein

expression

Activity assayby NMR(E. Coli UP in Cell

Lysate)


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


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


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

Parallel high throughput expression of Thermostable Phosphorylases Focus on technology

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