improved methods for fast analysis of producible ... · improved methods for fast analysis of...

Improved methods for fast analysis of producible

recombinant proteins in Hi5 insect cells

Maren Bleckmann, Margitta Schürig, Joop van den HeuvelRecombinant Protein Expression (RPEX)Helmholtz Centre for Infection Research

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Structure and Function of Proteins

Protein Sample Production Facility-PSPFRG-Recombinant Protein Expression

Helmholtz Centre for Infection ResearchBraunschweig, Germany

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• Fast• Inexpensive• High yields

Helmholtz Centre for Infection Research

• Not possible to produce all proteins

• No Glycosylation

• Not possible to produce all proteins

• Glycosylation pattern

• Simplest eukaryotic system

• Inexpensive• High yields

• Mammalian• Fast TGE

expression (Plasmid-based)

• High yields

• Stable• Time-intensive• Expensive• Glycosylation

pattern

• Mammalian• Long-time

expressionstable cell line

• Fast TGE optional

• Expensive• Time-intensive• “High” yields

(High copy cell lines)

• Low Yield(RMCE-single copy number)

E. coli Yeast HEK293-6E Sf9/Sf21/Hi5 CHO lec

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Role of Insect/BEVS in Recombinant Protein Expression

Page 4 |

Ø Eukaryotic system

Ø Serum free, high cell density and suspension culture

Ø Safety = Not human pathogenic

- Example: production of human vaccines

Ø Simple homogenous glycosylation pattern

- Advantages for crystallography

Source: PDB 06.11.2015

Baculoviral Expression Vector System I

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Bac-to-Bac™ (Invitrogen), Multibac, iACEMBL

Gene of interest

Tn7Tn7

Donor Transformation

Competent DH10BacTM E.coli Cells

lacZ

Bacmid

HelperBlue/WhiteSelection

RecombinantBacmid

Transformationof

Insect Cells

RecombinantVirus

VirusAmplification

Protein ProductionInfection of Insect Cells

Bacmid

lacZ orf1629orf603

Bsu36I Bsu36I Bsu36I

Gene of interest+orf603/1629

Donor

BakPAK6TM, Flashbac

a) Restriction with Bsu36I

b) Cotransfection of insect cells

RecombinantVirus

VirusAmplification

Protein ProductionInfection of Insect Cells

Baculoviral Expression Vector System II

Pro

Baculoviral System

Adv

anta

ges

Ø Very well established

Ø High yields

(especially for intracellular proteins)D

raw

back

s

Ø Time consuming

Ø Transient expression

Ø Lytic system

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Baculoviral System

Adv

anta

ges

Ø Very well established

Ø High yields

(especially for intracellular proteins)D

raw

back

s

Ø Time consuming

Ø Transient expression

Ø Lytic system (secreted protein)

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Contra

BaculoviralExpression

2-3weeks

72h

Challenge expressionGeneofInterest

Hugeworkloadandwhereistheprotein?

PlasmidbasedExpression

Maren Bleckmann |

LessWorkload !!!!!+PROTEIN

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Plasmid-BasedTransient Gene Expression

in Insect Cells

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Time after infection

Prom

oter

stre

ngth

Early

Late

Very Late(p10, polH)

Modified after King et al.

Host RNA Pol

ViralRNA Pol

Ø No host endogenous Sf21 promoters known so farØ Available promoters are restricted to EARLY viral promoters

Challenge: Choice of Promoters

GenomeSequencing

TranscriptomeSequencing

Data Alignment

Isolation of Putative

PromotersAnalysis of

Activity

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AAAAAA5` 3´

Strategy to identify endogenous Sf21 promoters

Ø Isolation of mRNA

Ø Sequencing six ScriptSeq RNA Libraries on the HiSeq2500

Ø Assembly of transcripts

Ø Transcript quantification with RSEM (RNA-Seq by Expectation Maximization)

High transcript level = strong promoter ?

Ø 30,405 transcripts with ORF >100bp

Ø Identification of 11,625 proteins with BLAST+

Strong analog promoter in other eukaryotic system = strong promoter ?

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GMAKRobert GeffersSabin BhujuMichael Jarek

Transcriptome Sequencing RNASeq

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Number of scaffolds (min 300bp) 51,304

N50 (bp) (QUAST 2.3) 133,811

Total basepairs 466,773,710

N's per 100 kb 3354.37

Largest scaffold (bp) 1,212,604

GC (%) 36.22

Complete ultra-conserved CEG hits (%) 99.19

Sf21 DNA was sequenced by Illumina sequencing technology and de-novo assembled

Draft genome of Spodoptera frugiperda published in August 2014 by Kakumani et al.

EMBL HeidelbergHüseyin Besir Markus H.-Y. FritzVladimir Benes

Sequencing results at a glance

De Novo Sf21 Genome Sequencing

Data Alignment

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ATG Intron Intron

AAAAAAAAAAAUG

5´ 3´

5´3´

TS

TS

Leader

Leader

Promising Upstream Region

~1000 bp

mRNA

DNA

Open Reading Frame

Open FrameReading

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Lipofectin®

Plasmid DNA

Insect Cells

Ø Transient plasmid transfection

Ø eGFP expression correlates with promoter activity

Ø Monitoring eGFP expression over 100 h

eGFP

PutativePromoter

Promoter Expression Analysis by TGE

17Maren Bleckmann |

Ø 48 well with max. 2 mL cell culture

Ø Shaking (400 – 1500 rpm; 3 mm orbital)

Ø Temperature (20oC- 50oC)

Ø Humidity (no/>80%)

Ø Online Biomass measurement (scattered light)

Ø Online GFP measurement (486 nm/510 nm)

High Throughput Analysis

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48 60 72 84

Tem

pera

ture

[°C

]R

elat

ive

Hum

idity

[%]

Hours after Transfection [h]

-100

100

300

500

700

900

1100

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48 60 72 84

Tem

pera

ture

[°C

]R

elat

ive

Hum

idity

[%]

Bio

mas

s G

ain

30


-100

100

300

500

700

900

1100

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48 60 72 84

Bla

nked

GFP

Gai

n 50


|

27°C

84%

Biomass BlankedGFP

Online Measurement in the Biolector

-100

100

300

500

700

900

1100

1300

1500

1700

1900

0 12 24 36 48 60 72 84

Bla

nked

GFP

Gai

n 50

Bio

mas

s G

ain

30


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Max. GFP Yield

Corrected eGFP yield

0

2

4

6

8

10

12

14

16

18

20

0 12 24 36 48 60 72 84 96

eGFP

Yie

ld

Time after Transfection [h]

0

2

4

6

8

10

12

14

16

18

20

0 12 24 36 48 60 72 84 96

eGFP

Yie

ld


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Ribos. 60S proteinRibos. C34 proteinRibos. S11 proteinRibos. L23A protein

EF-1alphaGAPDHEnolaseActinPGKHsp70

Early Viral:OpiE1 (OpMNPV)

Comparison of Promoter Activity in Sf21 cells

0

2

4

6

8

10

12

14

16

18

20

0 12 24 36 48 60 72 84 96

eGFP

Yie

ld


0

2

4

6

8

10

12

14

16

18

20

0 12 24 36 48 60 72 84 96

eGFP

Yie

ld


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Expression level of the viral OpiE2 promoter in Sf21 compared to Hi5 cells

~50x

169

8.261

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

SF21 OpiE2 Hi5 OpiE2

Max

eG

FP Y

ield

Max eGFP Yield for OpIE2 promoter in Hi5 50 times higher than Sf21

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Ribos. 60S proteinRibos. C34 proteinRibos. S11proteinRibos. L23A protein


OpiE1 (OpMNPV)

Ø GAPDH and Ribosomal promoter are stronger than the early viral promoter OpiE1

Results of the endogenous Sf21 promoters in cells

0

100

200

300

400

500

600

max

. GFP

Yie

ld

0

500

1000

1500

2000

2500

3000

3500

max

. GFP

Yie

ld

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OpiE1 (OpMNPV)hr5Ie1p10 (AcMNPV)


Ø hr5Ie1p10 has higher activity than all Sf21 promoters

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

max

. GFP

Yie

ld

Ø OpiE2 promoter best promoter by far

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OpiE1 (OpMNPV)hr5Ie1p10 (AcMNPV)OpiE2 (OpMNPV)


Comparison to the BEVS system

12

54

1

59

0 10 20 30 40 50 60 70 80

Plasmid based (hr5-OpiE2)

BEVS (MOI 2)

Plasmid based (OpIE2)

BEVS (MOI 2)

Hi5

Sf21

eGFP Yield [µg/106 cells]

Ø Volumetric yield of virus-free plasmid based expression ~50% of BEVS in Hi5

Cells continue to grow

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0

500

1000

1500

2000

2500

0 12 24 36 48 60 72 84 96

CorrectedeG

FPYield

Hoursaftertransfection[h]

eGFPYieldinTransientExpression

HEK293-6E(CMV)

Hi5(OpiE2)

Ø Max. eGFP yield in Hi5 cells 55% of max. eGFP yield in HEK293-6E cells

Comparison of the Hi5 to the HEK293-6E transient expression

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Improved Transient Gene Expression in Insect Cell by

Transactivation with baculoviralco-infection

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Ø Transactivation improves expression by inducing viral promoter activity

Boost transient gene expression in Sf21 by transactivation

-5000

0

5000

10000

15000

20000

25000

0 12 24 36 48 60 72 84 96 108 120

NFI

[rfu

]

Cultivation time [hpt] hr5-IE1-p10 Plasmid-based

hr5-IE1-p10 Transactivation

p10 (pTriEx) Transactivation

Phase I Phase II Phase III Phase IV

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Ø Promoter constructs tested for transactivation

Analysis of essential promoter elements for transactivation

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Ø Transactivation requires both hr5 and p10

Transactivation in Sf21

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Ø Best promoter for transactivation hr5-OpIE2-p10

Transactivation in Sf21

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Summary

Ø Transient plasmid-based expression (TGE) in Hi5 lead to over half of the

protein levels gained in BEVS or HEK293-6E system

Ø Compared to BEVS the Protein Quality might be improved and TGE is much

faster (72 h compared to 2-3 weeks)

Ø TGE in Hi5 results in a suitable glycosylation for crystallography and is

cheaper than the HEK293-6E system (media!)

Outlook

Virus-free expression in Insect Cells

Ø Further improvement of the TGE in Hi5 cells for direct large scale protein production

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SplitGFP Expression AnalysisFast and Reliable Screening

System in Insect Cells

splGFP11(=15AA)

splGFP1-10

Ø Noexpression=>NOGFP

SplitGFP detection system

TGEwithPlasmidCo-transfection

construct

Maren Bleckmann |

splGFP11(=15AA)

splGFP1-10

SplitGFP detection system

TGEwithPlasmidCo-transfection

construct

Ø Expressibleconstruct=>GFP

Maren Bleckmann |

construct

36

Semi-High Throughput with BioLector

Maren Bleckmann |

High Throughput GFP Expression Analysis

0

100

200

300

400

500

600

ND1

ND2

ND3

ND4

ND5

ND6

ND7

ND8

ND9

ND1

0ND1

1ND1

2ND1

3ND1

4ND1

5ND1

6ND1

7ND1

8ND1

9ND2

0ND2

1ND2

2ND2

3ND2

4ND2

5ND2

6ND2

8ND3

1ND3

2ND3

3ND3

4ND3

5ND3

6ND3

7ND3

8ND3

9ND4

0ND4

1ND4

2ND4

3ND4

4ND4

5Med

ium Hi5

mcherry

GFP1

-10

mcherryGF

P11

iFLpGF

P11

Max.SplitG

FPYield

MWmaxYield

Maren Bleckmann |

Example: NOD2 (Stefan Schmelz, Andrea Scrima, RG SBAU)

Max SplitGFP Yield =Blanked GFP Gain50 x 1000OD Gain30 x Transfection efficiency

Maren Bleckmann |

Example: NOD2

! ?

Plasmid based Expression

BEVSExpression

Ø TheSplitGFPSignalcorrelatestotheSOLUBLEproteinexpressioninBEVS

100kDa70kDa

25kDa

15kDa

10kDa

55kDa

35kDa

1.Ab: Mouse α

Strep

2.Ab:Rabbit α

Mouse-AP

Insoluble Fraction

100kDa70kDa

25kDa

15kDa

10kDa

55kDa

35kDa

Soluble Fraction

Maren Bleckmann |

Comparison to HEK293-6E cells

0

100

200

300

400

500

600

700

800

ND1

ND2

ND3

ND4

ND5

ND6

ND7

ND8

ND9

ND1

0ND1

1ND1

2ND1

3ND1

4ND1

5ND1

6ND1

7ND1

8ND1

9ND2

0ND2

1ND2

2ND2

3ND2

4ND2

5ND2

6ND2

8ND3

1ND3

2ND3

3ND3

4ND3

5ND3

6ND3

7ND3

8ND3

9ND4

0ND4

1ND4

2ND4

3ND4

4ND4

5Med

ium

negativ

eGF

P1-10

mcherry-…

iFlp-GFP11

Max.SplitG

FPYield

HEKInsekten

Ø TheSplitGFP ScreenisfunctionalinHEK293-6Ecells

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Fast splitGFP Screening System in Insect Cells

Summary

Outlook

Ø Fastandreliablescreeningofdifficulttoexpressconstructs

Ø RepresentssolubleexpressionandcorrelatestoyieldinBEVS

Ø Intotaltestedfor71differentconstructsof4targetproteins

Ø Functionalinothereukaryoticcells

Ø EstablishSplitGFPforlibraries(systemforstablecelllines)

Ø Establishdetectionsystemforsecretedtargetproteins

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Thank you ………..

RPEXJoop van den HeuvelSteffen MeyerBahar BaserChristian SchinkowskiMargitta SchürigJohannes SpehrNadine KonischAnke SamuelsDaniela GebauerKatharina Karste………

Students/InternsFang-Fang ChenDorina SchäckermannNils LindemannZen-Zen YenMargitta Schürig Jaqueline FrankeSelina SanderAngela BrandMargó PuschnerLara Ehemann

SBAUAndrea ScrimaStefan SchmelzStefan LeupoldPetra Völler…..

GMAKRobert GeffersSabin BhujuMichael Jarek

EMBL HeidelbergHüseyin BesirVladimir BenesMarkus H.-Y. Fritz

PSPFAnja SchützArie Geerlof

Literature

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Genomic analysis and isolation of RNA polymerase II dependent promoters fromSpodoptera frugiperdaMaren Bleckmann, Markus H.-Y. Fritz, Sabin Bhuju, Michael Jarek, Margitta Schürig, Robert Geffers, Vladimir Benes, Hüseyin Besir, Joop van den HeuvelPloS One; PMID: 26263512

Fast plasmid based expression screen in insect cells using SplitGFPMaren Bleckmann, Stefan Schmelz, Christian Schinkowski, Andrea Scrima, Joop van den HeuvelBiotechnology and Bioengineering; doi: 10.1002/bit.25956

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect CellsMaren Bleckmann, Margitta Schurig, Fang-Fang Chen, Zen-Zen Yen, Nils Lindemann, Steffen Meyer, Johannes Spehr, Joop van den HeuvelPlos One; doi:10.1371/journal.pone.0149424

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