TSB Q7 Meeting

02-Jun-2009

Hepatacore

iQur Leeds Progress

Overview

• Tandem core production» HA-tandem trials and tribulations

• Dual tandem production» eGFP-tandem» sAg,eGFP-dual tandem

• Space(r) Tandem core» HAVP1-tandem core» sAg-tandem core

• Future work

Bacteria expression

• Sizes of the tandems discussed:

• CoHo7e, HA1s (67kDa)• CoHo7e, eGFP (64kDa)• CoHo7sAg, eGFP (69kDa)• CoHo7e, sAg (spacers) (~43kDa)• CoHo7e, HAVP1 (spacers) (~128kDa)

Batch 7 HA – Tandem core purificationDiscontinuous sucrose gradient

Load

Pel

let

2 3 4 5 6 7 8 9 10 11 12 30M

Bottom Top

1ml fractions40%60% 30%

Load

Pel

let

2 3 4 5 7 8 9 10 11 126

Bio

tin M

r

Cores band at the 60:40% interface

Making HA-tandem… or not

• Adapted from the novel coHo7sAg purification method (see Q6 presentation), a similar high pH – low pH approach was attempted with HA-tandems

• Also attempted higher pH, lower buffer concentration

• Removal of incompatible reagents (Tween 20 precipitates with AmSO4)

• Increasing induction temperature to decrease heavy sediments (16 raised to 27oC)

An example of how NOT to do it…

2 3 4 5 6 7 8 9 10 11 31PM

Bottom Top

1ml fractions40%60% 30%

Eden cell pellet16oC inductionHigh pH lysis (pH8.5)=> Heavy sediment

2 3 4 5 6 7 8 9 10 11PLoadM

Bottom Middle

1ml fractions40%60%

iQur cell pellet27oC inductionNormal pH lysis (pH7.5)=> Lower overall yield

67kDa

HA-Tandem (+ fiends) shipped

HA-tandem

50

37

150

250

25

75

20

100

15

10

50

37

150

250

25

75

20

100

15

10

50

37

150

250

25

75

20

100

15

10

S/N PM

Lessons learnt for HA-tandem

• High pH adversely affects HA1 tandems core sedimentation

• Altered morphology can not be recovered by lowering pH

• Tween 20 is recommended at the start (post lysis) to prevent aggregates

• Low temperature expression levels yield better final product

» (less co-purifying ~37kDa contaminant, which is probably further reduced by differential precipitation with Ammonium Sulphate)

Green Dual tandem - Intro

• Dual construct needed to show “USP”

• eGFP correctly folded fluorescent activity present

• Easy to detect

• Both HBsAg and eGFP inserts have previously been shown to elicit immune responses

• Can be used for preliminary studies in non human primates

Dual tandem cloning

• pET28b-coHe7sAg,eGFP

Core II (aa1-185)

pET 28b-CoHe7e

HiseGFP

Core I (aa1-149)

Nco I Bam HI Not I Eco RI Xho ISac I Sal I

Flexible linker

Nhe

Core II (aa1-185)

pET 28b-CoHe7e,eGFP

His

Hetero tandem

Nhe I

pET 28b-CoHo7sAg,e

HissAg

Nco I Bam HI Not I Eco RI Xho ISac ISal I

Flexible linker

pET 28b-

His

Digest EcoRI/NheI

Homo tandem

Expression not optimal at 37 ˚C

M MNI

M M

I NI I

NI I NI I NI I

NI I

75kDa

25kDa

pET coHo7e

pET coHo7e,

eGFP

pET coHo7sAg,

eGFP pET coHo7e

pET coHo7e,

eGFP

pET coHo7sAg

,eGFP

75kDa

25kDa

75kDa

25kDa

Small scale expression

• Growth to an OD of 0.7 at 37˚C

• Induction with 1mM IPTG for 4h at 37˚C

60

40

30

cores

Analyses:BradfordSDS-PAGEWestern blotELISATEM

IPTG

French Press

14000 psi

Crude Dual tandem core prep

16oC

Sonicat

ion

Lysis in Tris pH8, 5% glycerol, 5mM DTT, Prot. Inhib, benzonase:

Clarification: 26k x g spin

Soluble

Insoluble

2 passesQuick

Pellet30%sucrose

+ 150mM NaCl

pET coHo7e,eGFP

pET coHo7sAg,eGFP

M NI I M NI I

M: Marker; NI: Non Induced; I: Induced

Expression

M T T M I S I S

250 150 100

75

50

37

25 20

10

15

pET coHo7e,eGFP

pET coHo7sAg, eGFP

M: Marker; T: Total Lysate; I: Insoluble Lysate; S: Soluble Lysate

Solubility

Most of the protein is soluble

Large scale prep (at 16oC)

250 150 100

75

50

250 150 100

75

50

M : Marker; L: Load; S/N: Supernatant; P : Pellet

M L L M S/N P S/N P

pETcoHo7e, eGFP

pETcoHo7sAg, eGFP

250

100 75

50

150

37

25 20

10

15

L M S/N P S/N P

250

100 75

50

150

37

25 20

10

15

M L

pETcoHo7e, eGFP

pETcoHo7sAg, eGFP

Western blot: 10E11 dil.1/4000; Goat anti mouse dil.1/2000M: Marker; L: Load; S/N: Supernatant; P: Pellet

Particulate material inferred by sedimentation

pETcoHo7e,eGFP and pETcoHo7sAg,eGFP Bradford assay

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20 25 30 35 40

Fractions

Co

nc

(mg

/mL

)

pETcoHo7e,eGFP

pETcoHo7sAG,eGFP

Discontinuous Sucrose GradientBradford assay

M 7 8 9 10 11 12L P 5 6 13 14

coHo7sAg, eGFP

M 7 8 9 10 11 12L P 5 6 13 14

coHo7e, eGFP

Discontinuous Sucrose GradientSDS-PAGE Coomassie stained gels

Broad range of sedimentation for both tandems

M 2 5 7 9 11 12 14 18 22 26 30 34

60% 40% 30%1ml fractions

Bottom TopBottom

coHo7sAg, eGFP (69kDa)

250150

10075

5037

2520

15

M 2 5 7 9 11 13 14 18 22 26 3430

1ml fractions60% 40% 30%

TopBottom

coHo7e, eGFP (64kDa)

250150

10075

50

37

2520

Antigenicity of gradient fractions e, eGFP (1)

pETcoHo7e, eGFP Disco. Gradient ELISA

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30 35 40

Fractions

Ab

sorb

ance

Anti sAg

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35 40

Fractions

Ab

sorb

ance

Anti eGFP

Anti core

Antigenicity of gradient fractions sAg,eGFP (2)

pETcoHo7sAg,eGFP Disco. Gradient ELISA

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30 35 40

Fractions

Ab

sorb

ance

Anti sAg

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35 40

Fractions

Ab

sorb

ance

Anti eGFP

Anti core

Preparation of pooled fractions

e, eGFP sAg, eGFP

50

37

150

250

25

75

20

100

15

10

S/N PM

50

37

150

250

25

75

20

100

15

10

S/N PM

Sucrose cushion pellet represents final preparation

Formulated in Tris (20mM, pH8) buffer with salt (150mM) and 40% sucrose

Major co-purifying contaminant at ~37kDa

sAg and HAVP1 space(r) types

Core I : atctggaaggatccgccggcggcggccgcgatccgg

L E G S A G G G R D P A

BamHI

Core II: gggtcggcaataatctggaattcgccggcgctagcgacEcoRI NheI

N L E F A G A S D P A S

NotI

Antigen insert site II

Nhe IEco RI

HAVP1

Afl II

TYPE AAntigen insert site II

Nhe IEco RI

HAVP1

KpnI

TYPE C

Antigen insert site II

Nhe IEco RI

HAVP1

KpnIAfl II

TYPE B

Antigen insert site II

Nhe IEco RI

sAg

Afl II

Antigen insert site II

Nhe IEco RI

sAg

KpnI

Antigen insert site II

Nhe IEco RI

sAg

KpnIAfl II

16C

Uni

nd T

ype

A

30C

O

/N T

ype

A

16C

Uni

nd T

ype

C

30C

Uni

nd T

ype

A

30C

Uni

nd T

ype

B

30C

Uni

nd T

ype

C

30C

O/n

Typ

e C

Mar

ker

16C

Uni

nd T

ype

B

16c

O/N

Typ

e B

16C

O/N

Typ

e C

16C

O/N

Typ

e A

30C

O/N

Typ

e B

25015010075

50

37

2520

128 kDa

Blot of HAVP1 Spacers expression at 16°C and 30°C

• Type A gives better expression than type B

• Type B produces more lower order species

• Type C least well expressed

• 16oC (weak but visible) better than 30oC (very faint) in all cases

150KDa

100KDa

16C

Tot

al

Mar

ker

Mar

ker

16C

Sol

uble

16C

Ins

olub

le

16C

Tot

al

16C

Tot

al

16C

Sol

uble

16C

Ins

olub

le

16C

Sol

uble

16C

Ins

olub

le

Type A Type B Type C

* * *

* Sample not correctly normalised (15-fold conc)

HAVP1 space(r) solubility shows little variation

• Solubility [more > less] follows [C > A > B]• Types A and C appear slightly more soluble than type B• Type A more abundant than the others

• Difficult to assess due to concentrated insoluble fraction and over exposure of blot

sAg spacer expression

M U 4 h

rs

O/N UU 4 h

rs

O/N

O/N

4 h

rs

M

50 KDa

37 KDa

Expression at 16 degree Celsius

42 KDa

Type A Type B Type C

Elute immobilised

fraction

VLPs

Immunoaffinity purification

• Possible generic purification approach capturing with anti-core

• Select for correctly folded inserts using conformation-dependent antibodies

• Typically low yields • Very expensive• Dependent on good

reagents

Wash off non-specific

contaminants

HCP

Load sampleImmobilise antibody

Future Work

• Optimise purification of dual tandem cores

• Set up immunoaffinity column

• Further expression and purification of space(r) constructs

• In vitro assays» Polyclonal anti-HAV» Alternative anti-HBsAg

• Engineering 4-helix bundle (?)