TSB Meeting 4

Hepatacore

iQur Leeds Progress

Overview

• Introduction

• CoHo7e,HA1s VLP purification

• Cloning

• Yeast cell lysis

• Future work

The tandem core platform

Core I (aa1-149)

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

Flexible linker

Antigen insert site I

Antigen insert site II

Nhe I

Core II (aa1-149)

pET 28b-CoHo7e

His

Homotandem core construct

Monomeric HBcAg (1-149)VLPs

Heterotandem HBcAg VLPs

60nM

Cryo-EM reconstructions of monomeric and tandem core particles. Performed by Dr R. Gilbert

(University of Oxford)

37 KDa

Tandem core proteinFlexible linker

Target Pathogens

Hepatitis B virus

• Enveloped virus

• Neutralising antigen surface antigen (HBsAg, aa124-137)

• Current vaccine – yeast expressed HBsAg VLPs

• 10 KDa insert

108155

Core I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I

HBsAg (108-155)Flexible linker

Antigen insert site I

Antigen insert site II

Target PathogensHepatitis A virus

VP

4 VP2 VP3 VP1

HAV P1

• Non-enveloped virus

• Neutralising antigen – cluster of epitopes in VP1 and VP3

• Current vaccines – live attenuated or inactivated whole virus

• 100 KDa insert

Core I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I

HAV P1 (aa1-791)

Flexible linker

Antigen insert site I

VP

4 VP2 VP3 VP1

135 KDa

Influenza Haemagglutinin

C2PR8HA_F2C2PR8HA_R2

C2PR8HA_F1C2PR8HA_R1

• H1 serotype (PR8) HA1 globular domain cloned into homo-tandem core

• Functional assay to confirm conformation of the haemagglutinin

• Protection studies can be done in a mouse model

• Express and purify from E.coli for optimisation assays

• Optimise haemagglutination and biophysical (EM, CD) assays

E.Coli expression and purification of CoHo7e,HA1s

• Resuspended sucrose cushion pellet is separated on a discontinuous sucrose density gradient (20-60%).

• A contaminating E.coli band (~ 37KDa) sediments with the CoHo7e,HA1s protein.

18211682

6449

37

26

1915

3 6 9 12 15 18 21 24 27 30 33 36M

Fractions collected from the bottom of the gradient

Removing the contaminant

Modification of sucrose density gradient purification• Heat treatment of lysate• Urea treatment of lysate

Affinity purification• His-tag nickel affinity column• Fetuin pull-down

Anion exchange chromatography• +/- 2M Urea• +/- Dialysis pretreatment

Ammonium sulphate precipitation

Final purification method

1. LysisFrench press: 14,000 psi, 2 passes.Sonication: 6 cycles 10s on, 20s off, 10 micron amplitude (on ice).Tween-20 treatment: 0.05% Tween-20 incubated at room temperature on rotating mixer for 1 hr.Clarification spin: Centrifugation of lysate at 50,000 x ‘g’ for 1 hr at 8 °C Dialysis: dialysed against 50 mM Tris HCl pH8.0, 2 M urea for 24hr at

4 °C using a 10 kDa MWCO Slide-a-Lyzer (Pierce) 2. Ammonium sulphate precipitation

50% ammonium sulphate added to the dialysed cell lysateOvernight incubation, protein and centrifugation at 26,000 x ‘g’ for 30 min

3. Discontinuous sucrose gradient30%, 40% and 60% sucrose in 20mM Tris HCl pH7.0 or pH8.0, 250 mM NaCl

4. Concentration and buffer exchangeCentriprep 10KDa filter

Analysis of CoHo7e,HA1s VLPs

M I 54 64

M I 54 6

pH 7.0 pH 8.0

CoHo7e,HA1s

SDS PAGE analysis of discontinuous sucrose density gradient purification of prep VIII CoHo7e,HA1s.

M. Mw markers, I. Input (resuspended ammonium sulphate precipitated protein)4-6. Sucrose gradient fractions 4-6 from the bottom of the gradient.

Analysis of CoHo7e,HA1s VLPs

2 M1

CoHo7e,HA1s

SDS PAGE analysis of CoHo7e,HA1s final sample.M. Mw markers,1. 1 μl sample, 0.5 μl

sample.

M 1 1M

CoHo7e,HA1s

A B

Western blot analysis of CoHo7e,HA1s final sample.

Gels. A: Anti-HBcAg (10E11); B: Anti-His tag. M. Mw markers, 1. final sample

Analysis of CoHo7e,HA1s VLPs

Anti PR8 and anti HBcAg ELISA

0

0.5

1

1.5

2

2.5

1 in 5

1 in 10

1 in 20

1 in 40

1 in 80

1 in 160

1 in 320

1 in 640

1 in 1280

1 in 2560

well

Ab

so

rba

nc

e

anti-PR8

anti-HBcAg

Anti-HBcAg (10E11) and anti-PR8 ELISA analysis serially diluted

CoHo7e,HA1s final sample

EM analysis of CoHo7e,HA1s final sample

Analysis of CoHo7e,HA1s VLPs

Haemagglutinantion assay of CoHo7e,HA1sSamples incubated with chick erythrocytes for 72hr at room temperature

PBS only

coHo7e,HA1s Prep X + Tween 20

coHo7e,HA1s Prep X - Tween 20

coHo7e,HA1s Prep XI – Tween 20 coHo7e Prep I – Tween 20

Influenza PR8

Transfer of constructs to yeast expression vector

• All constructs to be used in this study must be transferred to a Pichia pastoris expression vector

• pPICZ-C was the vector selected.

• For initial studies of tandem core expression in yeast, the following vectors were prepared

1. Wt HBc149 (to compare wt sequence with E.coli optimised sequences).

2. CoHo7e (empty E.coli optimised tandem core).

3. CoHo7e,eGFP (eGFP provides a simple assay for expression levels during optimisations).

4. CoHo7e,HA1s (Haemagglutinin construct).

Transfer of constructs to yeast expression vector

Following successful cloning of these constructs, SOPs were written

and carefully followed to avoid problems in future cloning steps

Transfer of constructs to yeast expression vector

The following constructs are now being prepared:

1. CoHo7e,HAVP1 (HAVP1 sequence in core II)

2. CoHo7,sAg (HBV surface antigen in core I)

3. CoHo7sAg,HAVP1 (dual inserts- sAg in core I and HAVP1 in core II)

More problems with cloning have delayed the production of these constructs!!!

Cloning Troubleshooting

A list of steps involved in the cloning process was drawn up.

1. Plasmid purification / PCR amplification of inserts

2. Restriction digestion

3. Gel purification of insert and vector

4. Ligation

The only change from previously optimised cloning experiments was found to be the use of a new UV light box!

Could the UV light now be damaging insert and vector?

A safe blue filter was used on the UV light box to limit damage of the DNA.

Cloning Troubleshooting

The following experiment was set up to determine the efficiency of plasmid digestion and re-ligation / transformation.

1. Digest plasmid with a pair of enzymes to release an insert

2. Heat inactivate enzyme

3. Ligate for 30 min / 1 hr at RT or 14°C (+/- ligase)

4. Heat inactivate ligase at end of incubation

5. Run ligation mixes on an agarose gel to check for re-ligation

6. Transform E.coli DH5α with ligated plasmid

Result:

Ligation and transformation were successful in the presence of ligase

Cloning Troubleshooting

30 60 30 60

RT 14oC

minsNo

Liga

se

Unc

ut

M

+ Ligase

Cloning Troubleshooting

A second experiment was set up to determine the effect of gel purification on ligation / transformation.

1. Digest plasmid with a single enzyme to linearise

2. Heat inactivate enzyme and gel purify half of the reaction

3. Ligate both gel purified and non purified linearised vector (+/- ligase)

4. Transform E.coli DH5α with ligated plasmid

Result:

Ligation and transformation were successful in the presence of ligase for both gel purified and non-purified linearised vector.

Cloning Troubleshooting

Cut vector – Ligase

Cut vector + Ligase

Gel purified Not Gel purified

4colonies

20colonies

~180colonies

>200colonies

Alternative yeast vector

One factor in the cloning difficulties has been the use of the pPICZ-C vector.

Many of the useful restriction sites are found within the vector making cloning strategies complicated

Zeocin selection (bacteriostatic) has led to screening of negative clones.

Expression of the constructs has not given high yield of protein

An alternative vector pPIC 3.5K has both ampicillin and kanamycin resistance markers.

Alternative yeast vector

Cloning pPIC3.5K-CoHo7e

Core I (aa1-149)

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

Flexible linker

Antigen insert site I

Antigen insert site II

Nhe I

Core II (aa1-149)

pPICZC CoHo7e

Xcm I

Kpn I

Xho I

PCR Amplify

Digest pPIC3.5K with EcoRI and Not I

Digest insert with EcoRI and Psp0MI

Eco RI Mlu IAfl IISpi I Psp0M I

Xcm I

pPIC3.5K CoHo-R1_3’

Not IEco RI Mlu IAfl II

Spi I

Psp0M I

Bam HI Not I Eco RISac I Sal IXcm I

Digest pPICZC with XcmI and EcoRI

Bam HI Not I Eco RISac I Sal I

pPIC3.5K CoHo7e

Mlu IAfl II Spi IXcm I

Clone into pPIC3.5KCoHo-R1_3’ (XcmI/EcoRI digested)

Cloning pPIC3.5K-CoHo7e

~450bp~250bp

coHo7e, e coHo7e, sAg

Yeast cell lysis

The following yeast cell pellets were obtained from Mologic

• Wt HBc149 (to compare wt sequence with E.coli optimised sequences) 18kDa.

• CoHo7e (empty E.coli optimised tandem core) 37kDa• CoHo7e,eGFP (65kDa)• CoHo7e,HA1s (67kDa)

Lysis method French press: 14,000 psi, 4 passes.Sonication: 6 cycles 10s on, 20s off, 10 micron amplitude (on ice).Tween-20 treatment: 0.05% Tween-20 incubated at room temperature on rotating mixer for 1 hr.Clarification spin: Centrifugation of lysate at 50,000 x ‘g’ for 1 hr at 8 °C

Yeast cell lysis

Western blot detection was necessary to detect any expressed protein

Only CoHo7e,eGFPs expression lysates gave positive results

It appeared that the core protein was soluble but insoluble material may not enter the gel

Glass bead vortexing did not improve lysis

PCR analysis of genomic DNA showed that each of the cell expression pellets were from transfected yeast cells

Use of a bead beater (3 min, 0.5mm beads) allowed detection of several constructs by western blot

Yeast cell lysis

SDS PAGE and western blot (anti-core) analysis of yeast cell lysis (CoHBc149 and CoHo7e,eGFP expression)

M T SI

CoHo7e eGFP

T SI

CoHBc149

MT SI

CoHo7e eGFP

T SI

CoHBc149

Yeast cell lysis

Western blot (anti-core) analysis of yeast cell lysates - bead beater lysis

Mco

Ho7

eeG

FP

HB

c14

9(B

1)

HB

c14

9(B

2)

coH

o7e,

HA

1s(F

1)

Tot Sol Tot Sol Tot Sol Tot Sol

coH

o7e,

HA

1s(F

2)

coH

o7e,

eLe

eds

(A1)

coH

o7e,

eLe

eds

(C1)

coH

o7e,

eM

olo

gic

(D1)

MTot Sol Tot Sol Tot Sol Tot SolMco

Ho7

eeG

FP

HB

c14

9(B

1)

HB

c14

9(B

2)

coH

o7e,

HA

1s(F

1)

Tot Sol Tot Sol Tot Sol Tot SolMco

Ho7

eeG

FP

HB

c14

9(B

1)

HB

c14

9(B

2)

coH

o7e,

HA

1s(F

1)

Tot Sol Tot Sol Tot Sol Tot SolMco

Ho7

eeG

FP

HB

c14

9(B

1)

HB

c14

9(B

2)

coH

o7e,

HA

1s(F

1)

Tot Sol Tot Sol Tot Sol Tot SolTot Sol Tot Sol Tot Sol Tot Sol

coH

o7e,

HA

1s(F

2)

coH

o7e,

eLe

eds

(A1)

coH

o7e,

eLe

eds

(C1)

coH

o7e,

eM

olo

gic

(D1)

MTot Sol Tot Sol Tot Sol Tot Sol

coH

o7e,

HA

1s(F

2)

coH

o7e,

eLe

eds

(A1)

coH

o7e,

eLe

eds

(C1)

coH

o7e,

eM

olo

gic

(D1)

MTot Sol Tot Sol Tot Sol Tot Sol

coH

o7e,

HA

1s(F

2)

coH

o7e,

eLe

eds

(A1)

coH

o7e,

eLe

eds

(C1)

coH

o7e,

eM

olo

gic

(D1)

MTot Sol Tot Sol Tot Sol Tot SolMTot Sol Tot Sol Tot Sol Tot Sol

CoHo7e,eGFPE1

CoHo7e,HA1sF1

HBc149B1

HBc149B2

CoHo7e,HA1sF2

CoHo7eA1

CoHo7eC1

CoHo7eD1

Expression of sAg in core I or core II (E.coli)

All current tandem core clones have foreign sequence inserted in core II

For production of a tandem core construct with two inserts, HBV surface antigen (sAg) was transferred to core I

pET28b CoHo7e was used as the parental vector

Expression of CoHo7sAg,e and CoHo7e,sAg was compared in E.coli BL21/DE3 cells

Expression of sAg in core I or core II (E.coli)

M U I U I

sAg in Core I

sAg in Core II

M

sAg in Core I

sAg in Core II

MU I U I

SDS PAGE and western blot (anti-core) analysis of CoHo7,sAg,e and CoHo7e,sAg expression in E.coli

Improved expression of sAg in core I !

Solubility of sAg in core I or core II (E.coli)

sAg in Core I

SDS PAGE analysis of CoHo7,sAg,e and CoHo7e,sAg solubility

Both proteins show good solubility

M T SI

+ Tween 20

T SI

-Tween 20

M T SI

+ Tween 20

T SI

-Tween 20

sAg in Core II

Future work

Cloning

1. Complete pPIC3.5K,CoHo7e

2. CoHo7e,sAg and CoHo7e,HAVP1 to pPIC3.5K vector (iQur/UoL)

3. Prepare dual insert construct (sAg ad HAVP1)

4. Optimisation of construct sequences for folding and solubility

Expression & purification of VLPs

1. Send above constructs pPIC3.5K constructs to Mologic for expression

2. Optimise bead mill lysis of yeast cell pellets

3. Optimise scalable purification processes