orip idea! - p4eu.org · page 1 p4eu meeting ׀ prague 2017 jvh joop van den heuvel, maren...

׀ 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!


Helmholtz Centre for Infection Research

Braunschweig, Germany

Recombinant Protein Expression


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)


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)


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


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.


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


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


High tdTomato Fluorescence

Binary-RMCE Cell Lines (SMT_dneo(2)_24 tdTomato)

Lower tdTomato Fluorescence


Binary cell lines tdTomato and double tdTomato


Expression of tdTomato is cumulative


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?


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


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)


GFP11 (= 15AA) +

GFP1-10

Insoluble construct => NO GFP

construct

Soluble construct => GFP

Split GFP Transient Transfection screen


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.


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)


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


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


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


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


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


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


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


Fraction of transfected cell

The pOpIE2-eGFP expression vector was used to analysis various

parameter on transfection efficiency in High Five cells


After adaptation for at least 3 months High Five cells performed best in

Excel 405 > Express Five> Sf900II

GFP Fluorescence (X-mean value)


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


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


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


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


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)

orip idea! - p4eu.org · page 1 p4eu meeting ׀ prague 2017 jvh joop van den heuvel, maren...

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