Improved methods for fast analysis of producible ... · Improved methods for fast analysis of...
Transcript of Improved methods for fast analysis of producible ... · Improved methods for fast analysis of...
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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
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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
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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
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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
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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
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BaculoviralExpression
2-3weeks
72h
Challenge expressionGeneofInterest
Hugeworkloadandwhereistheprotein?
PlasmidbasedExpression
Maren BleckmannPage 9 |
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
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GenomeSequencing
TranscriptomeSequencing
Data Alignment
Isolation of Putative
PromotersAnalysis of
Activity
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AAAAAA5` 3´
Strategy to identify endogenous Sf21 promoters
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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
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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
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17Maren BleckmannPage 17 |
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
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0
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0 12 24 36 48 60 72 84
Tem
pera
ture
[°C
]R
elat
ive
Hum
idity
[%]
Hours after Transfection [h]
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1100
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0 12 24 36 48 60 72 84
Tem
pera
ture
[°C
]R
elat
ive
Hum
idity
[%]
Bio
mas
s G
ain
30
Hours after Transfection [h]
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Bla
nked
GFP
Gai
n 50
Hours after Transfection [h]
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27°C
84%
Biomass BlankedGFP
Online Measurement in the Biolector
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-100
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0 12 24 36 48 60 72 84
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nked
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Gai
n 50
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mas
s G
ain
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Hours after Transfection [h]
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Max. GFP Yield
Corrected eGFP yield
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0
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eGFP
Yie
ld
Time after Transfection [h]
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eGFP
Yie
ld
Time after Transfection [h]
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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
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eGFP
Yie
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Time after Transfection [h]
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eGFP
Yie
ld
Time after Transfection [h]
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Expression level of the viral OpiE2 promoter in Sf21 compared to Hi5 cells
~50x
169
8.261
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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
EF-1alphaGAPDHEnolaseActinPGKHsp70
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
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max
. GFP
Yie
ld
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. GFP
Yie
ld
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Ribos. 60S proteinRibos. C34 proteinRibos. S11proteinRibos. L23A protein
EF-1alphaGAPDHEnolaseActinPGKHsp70
OpiE1 (OpMNPV)hr5Ie1p10 (AcMNPV)
Results of the endogenous Sf21 promoters in cells
Ø hr5Ie1p10 has higher activity than all Sf21 promoters
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0
1000
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max
. GFP
Yie
ld
Ø OpiE2 promoter best promoter by far
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Ribos. 60S proteinRibos. C34 proteinRibos. S11proteinRibos. L23A protein
EF-1alphaGAPDHEnolaseActinPGKHsp70
OpiE1 (OpMNPV)hr5Ie1p10 (AcMNPV)OpiE2 (OpMNPV)
Results of the endogenous Sf21 promoters in cells
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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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500
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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
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10000
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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
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splGFP11(=15AA)
splGFP1-10
Ø Noexpression=>NOGFP
SplitGFP detection system
TGEwithPlasmidCo-transfection
construct
Maren BleckmannPage 34 |
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splGFP11(=15AA)
splGFP1-10
SplitGFP detection system
TGEwithPlasmidCo-transfection
construct
Ø Expressibleconstruct=>GFP
Maren BleckmannPage 35 |
construct
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36
Semi-High Throughput with BioLector
Maren BleckmannPage 36 |
High Throughput GFP Expression Analysis
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5Med
ium Hi5
mcherry
GFP1
-10
mcherryGF
P11
iFLpGF
P11
Max.SplitG
FPYield
MWmaxYield
Maren BleckmannPage 37 |
Example: NOD2 (Stefan Schmelz, Andrea Scrima, RG SBAU)
Max SplitGFP Yield =Blanked GFP Gain50 x 1000OD Gain30 x Transfection efficiency
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Maren BleckmannPage 38 |
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
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Maren BleckmannPage 39 |
Comparison to HEK293-6E cells
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5Med
ium
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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
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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