Construction of Plasmids

&

Purification of Core-Haemagglutinin VLPs

Overview

• Introduction

• Cloning influenza haemagglutinin

• Transfer to Pichia vectors

• Expression of haemagglutinin construct in E.coli

• 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

The tandem core platform

Tandem HBV core - eBFP Assembled CoHe7e,eBFP VLPs

Core I Core II

Linker

eBFPAntigen insertion

sites

Single insertion into tandem coreCore I Core II

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

eBFPFlexible linker

Antigen insert site I

Antigen insert site II

Tandem core - eBFP,eGFP dimerAssembled

CoHe7L3eBFP,eGFP VLPs

Core I

Linker

Antigen insertion sites

Core II

eBFP

eGFP

Dual insertion into tandem core

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

eBFPFlexible linker

Antigen insert site I

Antigen insert site II

eGFP

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

Cloning HA1 (PR8)

• PR8 strain of influenza obtained from NIBSC

• The virus was bulked in MDCK cells

• Mouse infection studies have been completed and the system is ready for protection studies

• HA1 globular domain encoding RNA was reverse transcribed and amplified by PCR

• Several different strategies to clone the HA1 globular domain into the CoHo7e vector failed

Cloning pPICZ C constructs

•Cloning CoHo7e and wt HBc149 was also attempted.

•pPICZ vectors were obtained from Invitrogen and large scale plasmid preps performed.

•After several attempts, the cloning was not successful.

Cloning Troubleshooting

A list of possible reasons for unsuccessful cloning was drawn up.

1. Water supply – Building works adjacent to the lab had resulted in disruptions to the water supply that may have led to contamination. Cloning into commercial vectors may have been successful due to the inclusion of pure water in the cloning kits.

2. Suboptimal purification of vector and insert after digestion leading to contamination with impurities that inhibit ligation.

3. Problem with buffers used in electrophoresis of digested products for purification

4. Contaminated enzymes lead to improper overhangs for sticky end ligation

5. Incomplete digestion of vector

Getting the cloning back on track

• Firstly the CoHo7e,HA1s cloning was tackled.

• All plasmids involved in the cloning were prepared using an alternative large scale plasmid purification kit. Biorad Quantum Prep kit was used in place of Qiagen Midi/Maxi prep kits

• Three different approaches to cloning were employed.

• A wide range of insert:vector molar ratios and DNA concentrations were assessed.

Getting the cloning back on track

Cloning strategies

Core I (aa1-149)Nco I Bam HI Not I

Eco RIXho ISac I Sal I

Flexible linkerAntigen insert site I

Nhe I

Core II (aa1-149)

pET 28b-CoHo7e

Core I (aa1-149)Nco I Bam HI Not I Eco RI Xho ISac I Sal I

Flexible linkerAntigen insert site I

Nhe I

Core II (aa1-185)

pET 28b-CoHe7e,HA1s

Hi s

Hi s

HA1s

HAVP1

Exchange HA1s only Exchange

C-terminal region

Exchange HA1s + core-

coding sequence

Successful cloning strategies

Current cloning status

• Three individuals gained CoHo7e,HA1s clones in one week.

• Unusual molecular ratios proved successful eg 30:1 (insert:vector).

• Lower total DNA concentrations gave more positive colonies.

• A new SOP has been written to ensure that these conditions are followed in the future.

Transfer to pPICZ-C vector

• All constructs to be used in this study must be transferred to the Pichia pastoris expression vector pPICZ-C

• For initial studies of tandem core expression in yeast, the following vectors are also required:

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).

• Each of these constructs have now been made and sent to Mologic for transformation and expression.

Transfer to pPICZ-C vector

• The tandem core-haemagglutinin construct CoHo7e,HA1s transfer to yeast for expression and use in stability and protection studies.

• The HA1s insert has been successfully transferred into pPICZ-C coHo7e and sent to Mologic.

• 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)

E.Coli expression and purification of CoHo7e,HA1s

• Expression and purification in E.coli was attempted to provide VLPs for protection and stability studies.

• Initial expression studies showed that the CoHo7e,HA1s is expressed in E.coli and is partially soluble.

Un

ind

uce

d

Ind

uce

d

M To

tal l

ysa

te

Inso

lub

le ly

sate

So

lub

le ly

sate

Cu

shio

n S

/N

Cu

shio

n p

elle

t

75

50

37

25

Expression cell pellet lysed by French press, sonicated and treated with 0.05% Tween-20 for 1 hr before clarification by centrifugation at 50,000 x ‘g’.

Supernatant placed over 30% sucrose cushion and VLPs concentrated by centrifugation at 150,000 x ‘g’.

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

E.Coli expression and purification of CoHo7e,HA1s

• Fractions containing CoHo7e,HA1s are combined and concentrated by centrifugation

• The protein pellet is resuspended and centrifuged over a continuous sucrose density gradient (20-60%).

• The contaminating E.coli band sediments with the CoHo7e,HA1s protein.

182116

82

6449

37

26

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

Fractions collected from the bottom of the gradient

Removing the contaminant

• HBV core VLPs are resistant to up to 3M urea.

• If the contaminant is not as urea-stable it may be possible to denature the contaminant while retaining the VLPs

• The sucrose density gradient purified material was treated with 0,1,2 or 4M urea prior to separation on a 5ml continuous sucrose density gradient.

Urea treatment

Removing the contaminant

Urea treatment

CoHo7e, HA1s Prep 4 Urea Denaturation Trial Anti-Core ELISA

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 2 4 6 8 10 12 14 16 18

Fraction (From Top)

Ab

sorb

ance

0M Urea

1M Urea

2M Urea

4M Urea

Removing the contaminantUrea treatment

0M 2M

4M1M

1 2 3 4 5 6 7 8 10 11 12 13 14 15 169

1 2 3 4 5 6 7 8 10 11 12 13 14 15 169

1 2 3 4 5 6 7 8 10 11 12 13 14 15 169

1 2 3 4 5 6 7 8 10 11 12 13 14 15 169

Fractions collected from the top of the gradient

18211682

6449

37

26

182116826449

37

26

182116826449

37

26

182116826449

37

26

Removing the contaminant

• HBV core VLPs may be heated to 60°C without loss of integrity.

• Heating of the E.coli cell lysate causes precipitation of contaminating proteins.

Heat treatment

pH 7.5 pH 8.5 pH 9.5Tot Sol Tot Sol Tot SolM 1 2 3 4 5

1. Total lysate

2. Insoluble fraction

3. Soluble fraction

4. Sucrose cushion pellet

5. Sucrose cushion supernatant

182116826449

37

26

182116

82

6449

37

26

Removing the contaminant

• Fetuin is a serum protein that binds to haemagglutinin.

• Fetuin was coupled to CnBr activated sepharose and used to pull down the CoHo7e,VLPs (batch purification)

Affinity purification (Fetuin)

M 1 2 3 4 5 6 7 MM 1 2 3 4 5 6 7 M

1. Input2. Flowthrough3. Wash 14. 1.5M NaCl 15. 1.5M NaCl 26. 3M NaCl 7. Sepharose beads

18211682

6449

37

26

182116826449

37

26

Removing the contaminant

• Using surface charge characteristics of the VLPs, it may be possible to separate them from the E.coli contaminant.

Anion exchange chromatography

CIM monolith QA disk

Removing the contaminant

• Clarified cell lysate was bound to the column low salt (0.1M NaCl) conditions and eluted in 1M followed by 2M NaCl (stepwise elution).

Anion exchange chromatography

Inpu

t

FT

Was

hM 6 7 8 11 12 13 14 15 16 22 23

18211682

6449

37

26

1M NaCl 2M NaCl

Removing the contaminant

• Clarified cell lysate was bound to the column low salt (0.1M NaCl) conditions and eluted in 0.1- 1M NaCl gradient.

Anion exchange chromatography

Fractions viewed by EM

Removing the contaminantAnion exchange chromatography

18211682

6449

37

26

19

15

M Inpu

t

43 49 51 53 55 M 57 61 63 65 67 71

2M W

ash

FT

1

37 41FT

2

FT

3

Was

h

39 4547 59 69 83

Removing the contaminant

• At least 50% of the CoHo7e,HA1s protein is insoluble.

• An inclusion body preparation showed that insoluble core protein is present in inclusion bodies.

• Denaturing and renaturing experiments using urea, high pH and detergent are being done in an attempt to prepare VLPs from the inclusion body material

Purification of inclusion bodies

Future workCloning

1. Transfer CoHo7e,HA1, CoHo7e,sAg and CoHo7e,HAVP1 to pPICZc vector (iQur/UoL)

Expression & purification of VLPs

1. Further investigate the use of anion exchange purification and inclusion body protein refolding to produce CoHo7e,HA1s VLPs (iQur/UoL)

2. Express CoHo7e,HA1s in P. pastoris (Mologic) and purify the resulting protein (iQur/UoL)

3. Optimise expression of CoHo7e,eGFP in P. pastoris and compare localisation in yeast cells with CoHe7e,eGFP

4. Express CoHo7e and wt monomeric core in Pichia to compare expression (Mologic)

5. Purify CoHo7e and HBc149 core VLPs compare solubility

6. Express CoHo7e,HA1s in P. pastoris (Mologic) and purify the resulting protein (iQur/UoL)