Construction of Plasmids

&

Analysis of Yeast Lysates

Update

Overview

• Changes at iQur Leeds / University of Leeds

• The tandem core platform• Development of biophysical analysis

techniques• Cloning influenza haemagglutinin• Transfer to Pichia vectors• Analysis of yeast expression• Future work

Changes in Leeds

1. iQur (Leeds) has moved to the University of Leeds Bioincubator

Changes in iQur (Leeds)

2. iQur (Leeds) and University of Leeds groups have grown

Andrew Foster (Res Tech)UoL

Gheeta Bheeshmachar (RA)UoL

Anusha Panjwani (RA) – iQur Leeds

Ana Hildalgo (MSc Student) UoL

Aadil El-Turabi (Scientist) iQur Leeds

+ Sophie Maucaurant (Res Tech) - to join 2nd January –iQur Leeds

The tandem core platform

B-cell epitope

DNA binding

RNA bindingHBV core protein1 183

e174-89

α-helices

HBV core particle

HBV core protein

HBV core dimer

Eco RI Xho INco I

Antigen insert site

Nhe I

Core (aa1-185)

pET 28b-CoHBc185

His

Eco RI Xho INco I

Antigen insert site

Nhe I

Core (aa1-149)

pET 28b-CoHBc149

His

Antigen insertion sites

Monomeric HBV core – eBFP dimer

Assembled HBc185,eBFP VLPs

Core I Core II

eBFPHBV Core

Eco RI Nhe I Xho INco I

eBFP

Antigen insert site

Insertion into monomeric core

22 KDa

16 KDa

The tandem core platformTandem core constructs

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 (aa1-185)

pET 28b-CoHe7e

His

a) Heterotandem core

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

b) Homotandem core

Monomeric HBcAg (1-149)VLPs

Heterotandem HBcAg VLPs

60nMCryo-EM reconstructions of monomeric and

tandem core particles. Performed by Dr R. Gilbert (University of Oxford)

42 KDa 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

Tandem core construct expression

The tandem core platform

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Codon Optimisation

• Constructs with the pre-fix ‘Co’ are codon optimised for expression in E.coli.

• Expression of the codon optimised sequence improved yields in E.coli by ~100x

• E.coli optimised sequences contain codons that are rarely used in P. pastoris

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

Biophysical Analysis

Analysis of VLPs has been undertaken as an MSc student project

Initially, monomeric full length (CoHBc185) and truncated core (CoHBc149) have been studied

UV scan analysis and EtBr stain agarose gel electrophoresis of VLPs show the increased nucleic acid binding of the full length core protein

CoHBc149

CoHBc149 CoHBc185

CoHBc185

Biophysical Analysis

Conclusions:

VLPs containing the C-terminal region of HBV core protein package high quantities of nucleic acid

This is an undesirable characteristic for VLP based vaccines.

All future work in this project is to be done with the Homotandem core (CoHo7e)

Cloning ProgressHepatitis B virus Surface Antigen

• HBsAg cloned into homotandem core insertion site II

• Transferred HBsAg sequence into homotandem core insertion site I

• Sequences have been analysed and are correct.

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

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

Cloning ProgressHepatitis A virus

• HAV P1 cloned into each empty tandem core construct:-

• Sequences are correct

• Low levels of expression in E. coli

• Protein does not appear to be at all soluble

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)-1

C2PR8HA_F2C2PR8HA_R2

C2PR8HA_F1C2PR8HA_R1

Influenza Haemagglutinin, an alternative insert for stability studies

• Previous E.coli expressed constructs fold well

• Functional assay to confirm conformation of the haemagglutinin

• Protection studies can be done in a mouse model

• H1 serotype HA1 globular domain cloned into each of the core constructs

• Sequencing confirmation required

• Express and purify from E.coli for optimisation assays

• Optimise haemagglutination and biophysical (EM, CD) assays

• Transfer to Pichia vectors and express

• Purify VLPs

• Set up stability study & ongoing analysis

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

Cloning HA1 strategy 1

• PCR primers encode restriction sites for direct cloning into prepared tandem core vector

Cloning HA1 strategy 1

• PCR primers encode restriction sites for direct cloning into prepared tandem core vector

EcoRI/NheI cut CoHo7e

EcoRI/NheI cut HA1 PCR product

CoHo7e,HA1

Cloning HA1 strategy 1

• PCR primers encode restriction sites for direct cloning into prepared tandem core vector

• No positive clones obtained despite several attempts by 3 different operators

Cloning HA1 strategy 1

• PCR primers encode restriction sites for direct cloning into prepared tandem core vector

• No positive clones obtained despite several attempts by 3 different operators

Why?Restriction digestion of PCR products or vector not complete?

Cloning HA1 strategy 2

• Directly clone undigested HA1 PCR product into commercial PCR cloning vector TOPO-pCRblunt (Invitrogen)

• Several positive colonies obtained

• Restriction digestion of HA1 insert to release from the pCRblunt vector and transfer to digested CoHo7e vector

• 4 positive clones obtained

• Each clone contained sequence carried over from the PCRblunt vector.

Cloning HA1 strategy 2

Why?

EcoRI restriction digest sites within the pCRblunt vector were preferentially cut whereas the EcoRI site within the insert was not cut.

Insert EcoRI site

Vector EcoRI site

Cloning HA1 strategy 3

• Directly clone undigested HA1 PCR product into alternative commercial PCR cloning vector pJET blunt (Fermentas)

• pJET blunt has no NheI or EcoRI restriction sites so only those in the insert will digest

• Several positive colonies obtained – sequence correct

• Restriction digestion of HA1 insert to release from the pCRblunt vector and transfer to digested CoHo7e vector

• 2 positive clones obtained as analysed by digestion

• Each clone contained totally unrelated sequence

Cloning HA1 strategy 3

Why?

Unknown contaminating plasmid transformed the E.coli

Expected restriction digest pattern obtained with several different enzymes coincidental!

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

Troubleshooting

1. Contaminated water supply

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

3. Faulty buffers used in electrophoresis of digested products for purification

4. Contaminated enzymes

These possible causes were investigated

• A previously digested and purified CoHe7e vector was used

• Ligation of the insert purified from pJETblunt resulted in a positive colony

• This clone was successfully sequenced

• None of the above factors are the cause of the cloning problem as each were involved in the preparation of the insert

• This is confirmed by the lack of improvement with alternative water and buffers.

Troubleshooting

5. Incomplete digestion of the vector

Investigation

• CoHo7e vector containing the HAVP1 seqeunce was purified

• Digestion of this insert results in the release of an insert that can be clearly seen on an agarose gel and a reduction in size of the vector such that only fully digested vector is excised and purified from the gel

• Cloning into this cut CoHo7e vector was unsuccessful in the first attempt.

• Further insert is now being prepared to retry ligation at various vector / insert ratios

Cloning CoHo7e into pPICZc and pPICZc-α

• The pPICZc and pPICZc-α constructs prepared using a large scale kit

• Digestion of pPICZc gave an expected 3Kb linearised vector

• Digestion of pPICZc-α gave a 6Kb linearised vector (3.6KB expected

• Very high yields of wt core sequence in Pichia have been reported (B. Watelet et al. / Journal of Virological Methods 99 (2002) 99–114)

• Since the clones that we have were not expressing to such high levels and one of the parental vectors are questionable, we purchased new vectors to clone

• As a control we attempted to clone HBc149 (wt core) into the pPICZc vector

• Homotandem core CoHo7e is also being transferred

• After several attempts, the cloning has not been successful as yet

How do we get past this bottleneck?

• Use alternative large scale plasmid purification kit. The only difference between the CoHe7e and the CoHo7e vector, is the purification kit used

• Alternative cloning strategy using In-Fusion system which is based on recombination rather than ligation

• Subcontract cloning of the critical constructs now and continue to troubleshoot the general cloning issue.

Detergent treatment of yeast lysates 1

• Pichia pastoris expressing CoHe7e (heterotandem core) were lysed by French press, sonicated and treated with various concentrations of Tween-20 for 1 hr before clarification by centrifugation at 50,000 x ‘g’

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Coomassie Blue Stained SDS PAGE gel

M 1 2 3 4 5 876 109M. Protein standards1. Whole cell lysate2. French press lysate3. 0% Tween-20 S/N4. 0% Tween-20 Pellet5. 0.01% Tween-20 S/N6. 0.01% Tween-20 Pellet7. 0.1% Tween-20 S/N8. 0.1% Tween-20 Pellet9. 1% Tween-20 S/N10.1% Tween-20 Pellet

Detergent treatment of yeast lysates 2

• Pichia pastoris expressing CoHe7e (heterotandem core) were lysed by glass bead vortexing, sonicated and treated with various concentrations of Tween-20 for 1 hr before clarification by centrifugation at 50,000 x ‘g’

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Coomassie Blue Stained SDS PAGE gel

M 1 2 3 4 5 876 109 M. Protein standards1. Whole cell lysate2. French press lysate3. 0% Tween-20 S/N4. 0% Tween-20 Pellet5. 0.01% Tween-20 S/N6. 0.01% Tween-20 Pellet7. 0.1% Tween-20 S/N8. 0.1% Tween-20 Pellet9. 1% Tween-20 S/N10.1% Tween-20 Pellet

Mini gradient analysis of detergent treated lysates.

• Clarified, Tween-20 treated lysates (10μl) were loaded on miniature (200μl) discontinuous gradients (20,40,60% sucrose) and centrifuged at 150,000 x ‘g’ for 30 minutes. Fractions were analysed by ELISA.

ELISA of Mini Gradient Fractions: Analysis of French Press Lysis of Yeast Expressed coHe7e Solubilized with Tween 20

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Expected sedimentation of HBV core VLPs

Isopropanol treatment of yeast cell lysates.

• Clarified, Isopropanol (+/- 0.05% Tween-20) treated lysates (10μl) were loaded on miniature (200μl) discontinuous gradients (20,40,60% sucrose) and centrifuged at 150,000 x ‘g’ for 30 minutes. Fractions were analysed by ELISA.

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ELISA of Mini Gradient Fractions: Analysis of French Press Lysis of Yeast Expressed coHe7e Solubilized with Isopropanol

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A. Without Tween-20 B. With 0.05% Tween-20

Large scale lysis of the yeast expressed CoHe7e protein

• Both Tween-20 and Isopropanol treated CoHe7e sediment as VLPs within the discontinuous sucrose density gradient. But yields are too low for continued purification.

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Expected sedimentation of HBV core VLPs

• Tween-20 treatment of lysates appears to release more VLPs than Isopropanol

Future work

Cloning

1. Influenza HA1 to be placed in CoHo7e (iQur/UoL)

2. CoHo7e and wt monomeric core (HBc149) to be placed in pPICZc vector (iQur/UoL)

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

Expression & purification of VLPs

1. Express CoHo7e,HA1s in E.coli and purify VLPs for use in stability and protection studies (iQur/UoL)

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

3. Purify CoHo7e and HBc149 core VLPs compare solubility

Future workDevelopment of Scalable Purification methods

• Currently using ultracentrifugation techniques extensively. These are time consuming and not practically scalable.

• Chromatography techniques have been slow – low flow rates required

• Gel filtration does not retard VLP sufficiently from void volume

• Monolith ion exchange matrix technology will be assessed in January

Biophysical Characterisation

• Analysis of tandem core VLPs will be initiated in January

• The following techniques will be done:

•Analylitical ultracentrifugation, EM, Circular Dichroism (particle characteristics)

•UV scan, Electrophoresis (Nucleic acid content