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Page 1 ׀ P4EU Meeting Prague 2017 JVH
Joop van den Heuvel, Maren Bleckmann, Margitta Schürig
Transient Gene Expression or what Hi5 insect cells
can mean for you
RG Recombinant Protein Expression and
Protein sample production facility (PSPF)
Promoter- region
Tn7 L
FRT3
FRTwt
GmR
Amp
MCS / GOI
SV40 pA
Tn7 R
PGK-Promoter
pUC ori
pFlp-Bac-to-Mam II
EBV oriP Idea!
Page 2 ׀ P4EU Meeting Prague 2017 JVH
Helmholtz Centre for Infection Research
Braunschweig, Germany
Recombinant Protein Expression
Page 3 ׀ P4EU Meeting Prague 2017 JVH
Recombinant Protein Expression Activities
Department of Structure and Function of Proteins at
Helmholtz Centre of Infection Research
Helmholtz Protein Sample Production Facility (www.pspf.de)
Instruct-ERIC - Integrated Structural Biology Infrastructure for Europe
Centre for Protein Production (www.structuralbiology.eu)
Protein Production and Purification Partnership in Europe
Network, Training, Exchange, Symposia
NEW!!! CSSB Hamburg at DESY Campus (PP@CSSB)
Page 4 ׀ P4EU Meeting Prague 2017 JVH
Platform Support: What do we offer?
Scientific Cooperation
Support to get protein samples for your project
Advice and Materials for do-it yourself cloning
Protein sample production
Entry requirement – Expression clone
Insect cell expression (BEVS)
Mammalian cell expression (CHO, HEK 293-6E)
Optionally Yeast (Pichia pastoris)
High Throughput Cloning by SLIC technology (KBU, DAG, WBL)
Page 5 ׀ P4EU Meeting Prague 2017 JVH
Production of soluble mammalian protein and
protein complexes
Structural analysis of receptor-ligand complexes
Host-Pathogen interaction
Functional proteomic studies
Complex biologics
Drug target protein
Proteins for new strategies in vaccinology
Page 6 ׀ P4EU Meeting Prague 2017 JVH
Transfection Antibiotic selection
Colonies
I. Development of a FACS-based selection
method to isolate high producer mammalian
cell lines for structural biology
FACS-sorting of GFP-transfected cells
Avoids antibiotic selection
Selection for strong, stable GFP
expression
Long-time Stability of selected
expression cell lines
Wilke et al. (2010) Prot. Sci.
Page 7 ׀ P4EU Meeting Prague 2017 JVH
Stable Cell Line Development using
Recombinase-mediated cassette exchange (RMCE)
From master to producer
cell line in less than 2
months
Random integration
Screening for stable clones
Targeting with pFlpBtM-vector
Screening for non-fluorescent
G418 resistant cells
eGFP Δneo EF
Promoter Production cell
FRT3 FRTwt
neoATGGOIEF
Promoter
PGK
Promoter
eGFP master cell
FRT3
eGFP
FRTwt
ΔneoEF
Promoter
FRT3
ATGGOI
FRTwt
FLP
PGK
Promoter
pFlpBtM
Donor vector
Wilke et al. PLoS One 6 (2011): e27829
FRT3 FRTwt
Page 8 ׀ P4EU Meeting Prague 2017 JVH
PEF FRT3 eGFP FRTwt Δneo
FRT3 1st GOI PGK Promoter ATG FRTwt
PEF FRT3 1st GOI PGK Promoter ATG FRTwt Δneo
PEF FRT13 tdTomato FRT14 Δpuro
FRT13 2nd GOI PGK Promoter ATG FRT14
PEF FRT13 2nd GOI PGK Promoter ATG FRT14 Δpuro
Genomic Locus 1: eGFP Genomic Locus 2: tdTomato
Essentials for Multi RMCE Cell Line Development
• New Fluorescent gene tdTomato
• New Set of Frt sites F13 - F14
• New Exchange Vector F13 – F14 Polylinker
• New Selection trap Δpuro
• New Cell Line Multi RMCE
Page 9 ׀ P4EU Meeting Prague 2017 JVH
High tdTomato Fluorescence
Binary-RMCE Cell Lines (SMT_dneo(2)_24 tdTomato)
Lower tdTomato Fluorescence
Page 10 ׀ P4EU Meeting Prague 2017 JVH
Binary cell lines tdTomato and double tdTomato
Page 11 ׀ P4EU Meeting Prague 2017 JVH
Expression of tdTomato is cumulative
Page 12 ׀ P4EU Meeting Prague 2017 JVH
Conclusion:
Stable binary RMCE Master cell lines
Long term stable expression without selection pressure
Expression level specific for each locus
Problem:
Still Time Consuming - Not suitable for Construct
Screening
How do we create a compatible HTP expression screen?
Page 13 ׀ P4EU Meeting Prague 2017 JVH
Baculoviral Expression
4 w
eeks
Plasmid based Expression
72
h
476,2
24,8
0
100
200
300
400
500
600
BEVS PlasmidTransfection
µg
eG
FP
yie
ld/
10
6cells
Construct screening in insect cell lines
13
Page 14 ׀ P4EU Meeting Prague 2017 JVH
The BioLector microcultivation system for HTP
expression screening
48 well with max. 2 mL cell culture
Shaking (400 – 1500 rpm; 3 mm orbital)
Temperature (20oC- 50oC)
Humidity (no/>80%)
Online Biomass measurement (scattered light)
Online GFP measurement (486 nm/510 nm)
Page 15 ׀ P4EU Meeting Prague 2017 JVH
GFP11 (= 15AA) +
GFP1-10
Insoluble construct => NO GFP
construct
Soluble construct => GFP
Split GFP Transient Transfection screen
Page 16 ׀ P4EU Meeting Prague 2017 JVH
Experimental set up for target NOD2
Coexpression of GFP1-10 and GFP11-NOD2 construct
Transfection: 0,5*106 cells/mL, Hi5-cells, 2 mL 2 µg DNA/well 1:2 DNA/Lipofectin Ratio
Cultivation at 900 rpm, 27oC, 96 h
splGFP1-10 OpiE2 Ie1 term
OpiE2 Ie1 term splGFP11 NOD2 const.
Page 17 ׀ P4EU Meeting Prague 2017 JVH
NOD2 constructs
17
0
100
200
300
400
500
600
ND
1
ND
2
ND
3
ND
4
ND
5
ND
6
ND
7
ND
8
ND
9
ND
10
ND
11
ND
12
ND
13
ND
14
ND
15
ND
16
ND
17
ND
18
ND
19
ND
20
ND
21
ND
22
ND
23
ND
24
ND
25
ND
26
ND
28
ND
31
ND
32
ND
33
ND
34
ND
35
ND
36
ND
37
ND
38
ND
39
ND
40
ND
41
ND
42
ND
43
ND
44
ND
45
GFP
1-1
0
mch
erry
GFP
11
iFLp
GFP
11
Max
. GFP
yie
ld
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) also known as caspase recruitment domain-containing protein 15 (CARD15)
Page 18 ׀ P4EU Meeting Prague 2017 JVH
25kDa
15kDa
10kDa
70kDa
35kDa
50kDa
Ma
rke
r
ND
1
ND
3
ND
4
ND
6
ND
8
ND
9
ND
17
ND
25
ND
43
Purification of 9 different Strep tagged NOD constructs in BEVS (15*106cells)
19 µg/106cells
Correlation of Fluorescence to expression
18
6,5 µg/106cells
Page 19 ׀ P4EU Meeting Prague 2017 JVH
Result:
Transient Gene Expression in Insect Cells (TGE)
Split GFP allows detection of Expression
Biolector System for HTP expression screen
Option 1:
Direct transfer into Baculo Viral Expression System
Option 2:
Generate RMCE Production Cell Line with optimized Construct
Option 3:
Scale up of TGE in Hi5
Page 20 ׀ P4EU Meeting Prague 2017 JVH
0
2
4
6
8
10
12
14
16
18
20
0 12 24 36 48 60 72 84 96
eG
FP
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
eG
FP
Yie
ld
Time after Transfection [h]
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
Ma
x e
GF
P Y
ield
Max eGFP Yield for OpIE2 promoter in Hi5 50 times higher than Sf21
Page 21 ׀ P4EU Meeting Prague 2017 JVH
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
Sf2
1
eGFP Yield [µg/106 cells]
Volumetric yield of plasmid based expression ~50% of BEVS in Hi5
Cells continue to grow
Page 22 ׀ P4EU Meeting Prague 2017 JVH
0
500
1000
1500
2000
2500
0 12 24 36 48 60 72 84 96
Co
rrec
ted
eG
FP Y
ield
Hours after transfection [h]
eGFP Yield in Transient Expression
HEK293-6E (CMV)
Hi5 (OpiE2)
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
Page 23 ׀ P4EU Meeting Prague 2017 JVH
Optimized and Simple Protocol for Transient Gene Expression in Hi5
Harvest exponentially growing High Five cells and resuspend at 5 x 106 cells/ml
Add 1 µg pOpIE2-eGFP plasmid for each 106 cells. Mix
Directly add PEI in a ratio of 4:1 to DNA. Mix and incubate for 3 hr (160 rpm, 27oC)
Dilute with fresh medium to 1 x 106 cells/ml and continue incubation (130 rpm, 27oC)
Follow Expression by flow cytometry
Harvest exponentially growing High Five cells
Resuspend at 5 x 106 cells/ml
Add 1 µg pOpIE2-eGFP plasmid for each 106 cells. Mix
Directly add PEI in a ratio of 4:1 to DNA. Mix and incubate for 3 hr (160 rpm, 27oC)
Dilute with fresh medium to 1 x 106 cells/ml and incubate (130 rpm, 27oC)
Follow Expression by flow cytometry
Serval different
Expression vectors
Protein
Production
Analysis in
Western Blot
PEI Transfection
100kDa 70kDa
25kDa
15kDa
10kDa
35kDa
55kDa
Serval different
Expression vectors
Protein
Production
Analysis in
Western Blot
PEI Transfection
100kDa 70kDa
25kDa
15kDa
10kDa
35kDa
55kDa
Serval different
Expression vectors
Protein
Production
Analysis in
Western Blot
PEI Transfection
100kDa 70kDa
25kDa
15kDa
10kDa
35kDa
55kDa
Page 24 ׀ P4EU Meeting Prague 2017 JVH
Growth rate after adaptation for at least 3 months to different media
Doubling time varies from 15 to 17 hours
Influence of Cell Line, Growth and Media on TGE
Page 25 ׀ P4EU Meeting Prague 2017 JVH
Fraction of transfected cell
The pOpIE2-eGFP expression vector was used to analysis various
parameter on transfection efficiency in High Five cells
Page 26 ׀ P4EU Meeting Prague 2017 JVH
After adaptation for at least 3 months High Five cells performed best in
Excel 405 > Express Five> Sf900II
GFP Fluorescence (X-mean value)
Page 27 ׀ P4EU Meeting Prague 2017 JVH
Effect of Bacviral DNA Sequences on Transient Gene Expression in Hi5
eGFP-HA,
eGFP-HA D 603
eGFP-HA D 1629
eGFP-HA D 603 D 1629
Page 28 ׀ P4EU Meeting Prague 2017 JVH
1 2 4 5 6 8 7 3 9
Lane Sample Amount
1 Precision Plus –unstained- (Biorad) 10 µl
2 Lysat 15 µl
3 DL 15 µl
4 A1 15 µl
5 B8 15 µl
6 B9 15 µl
7 B10 15 µl
8 B11 15 µl
9 B12 15 µl
10 C1 15 µl
SDS-gel / 12 %
Instant Blue
size of the protein: 57,8 kD
Intracellular protein
Sample: 4µl 4xLLPP + 15µl Probe
cell lysis: 20mM HEPES pH 8,0; 500mM NaCl; 1mM EDTA;
10% v/v Glycerin; 2mM TCEP; cOmplete-Mini;
Benzonase; 0,5% NP40
Remark: Pool: B9 – C1
Dialyse / Waschpuffer pH 7,4 / 6-8000MWCO /
5L/ 4°C
10
75
50
37
25
20
15
kD
P58
20.03.2017
SDS-gel
594
PSPF-Nr. HZI 53
P58 1-488_c-term Strep / OP 228
ÄKTA-FPLC / 1ml StrepTactin Superflow Cartridge (IBA)
TGE/ Hi5 /1L Culture / asa
15,8g Pellet / 80% Transfectionsefficiency (eGFP Reporter)
Project 53 Intracellular P58 protein
Page 29 ׀ P4EU Meeting Prague 2017 JVH
1 2 4 5 6 8 7 3 9
Lane Sample Amount
1 Precision Plus -unstained- (Biorad) 10µl
2 DL 10µl
3 Wasch 3 10µl
4 Elu 1 10µl
5 Elu 2 10µl
6 Elu 3 10µl
7 Elu 4 10µl
8 Elu 5 10µl
9 Elu 7 10µl
SDS-gel / 12 %
Instant Blue
size of the protein: 62 kD
Intracellular protein
sample: 4µl LLPP + 15µl sample
cell lysis: 0,5% Igepal
remark: Pool E1 – E7 (1,0mg/mlkorr.) in ca. 11ml [11,1mg total]
Dialyse (50mM Tris/HCl pH 8,0; 500mM NaCl;
10% v/v Glycerol; 2mM TCEP)
5L / 4°C / ON MWCO 6-8000
Superdex 200 26/60
75
50
37
25
20
15
kD
P35
05.05.2017
SDS-gel
606
PSPF-Nr. 41 int
P35_NStrep / OP 276
1,5ml StrepTactin Superflow (IBA)
MobiCol / Batch 500ml Lysat / ON
TGE / Hi5 /1L Culture / asa
25g Pellet / 56% Transfectionsefficiency (eGFP Reporter)
Project 41 Intracellular P35 protein
Page 30 ׀ P4EU Meeting Prague 2017 JVH
A Versatile Integrated Recombinant Expression Platform
First: Transient Transfection Screen with Split GFP Analysis
Second: Large scale Production
Stable cell line (CHO)
Baculo Viral Expression (Hi5)
NEW: Scale-Up TGE in Hi5 Insect cells in shake flask up to 1L
Further optimization of TGE in Hi5 for secreted protein is ongoing
Page 31 ׀ P4EU Meeting Prague 2017 JVH
Many thanks to: Recombinant Protein Expression (PSPF@HZI)
Margitta Schuerig
Volker Jäger
Daniela Gebauer
Nadine Konisch
Anke Samuels
Claudia Wylegalla
Former PhD Students
Sonja Wilke
Steffen Meyer
Bahar Baser
Maren Bleckmann
SFPR
Wulf Blankenfeldt
Konrad Büssow
Peer Lukat
Stefan Schmelz
Andrea Scrima
PSPF-Berlin-MDC
Anja Schütz (PSPF@MDC)
Arie Geerlof (PSPF@HMGU)
CSSB-Hamburg
Michael Kolbe (PP@CSSB)
Page 32 ׀ P4EU Meeting Prague 2017 JVH
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