beyond viagra: novel use of nus-a fusion and gateway ... · gateway cloning technology in the...

Beyond Viagra: Novel use of Nus-A fusion and Gateway cloning technology in the

heterologous expression of Plasmodium falciparum phosphodiesterases

Daniel T Leung, MD; Paul S Pottinger, MD; Wesley C Van Voorhis, MD, PhD

Division of Allergy and Infectious Diseases, Department of Internal Medicine, University of

Washington, Seattle, WA

Malaria

• Caused by infection by Plasmodiumspecies

• 300 million cases per year• Directly causes 1 million deaths per year• $12 Billion lost revenue in Africa

We need better anti-malarials!

• Increasing drug resistance• Poor efficacy of current medications

Does the answer lie in inhibitors of phosphodiesterases (PDE)?

Where have I heard ofPhosphodiesterases (PDE)?

• Enzymes involved in intra- and inter-cellular signaling

A

Ade

nyly

lC

ycla

se

GG

ATP

cAMPcAMP

PDEPDEPDEPDE

AMPG

uany

lyl

Cyc

lase

B

GTPcGMP

PDEPDE

GMPG

uany

lyl

Cyc

lase

B

GTPcGMPcGMP

PDEPDEPDEPDE

GMP

casc

ade

of b

ioch

emic

al re

actio

ns(C

a++

depe

nden

t PK

A, o

ther

s)

No, really… Where else have I heard of

Phosphodiesterases (PDE)?

PDE & Malaria

• 4 PDE genes exist in P. falciparum, functions unknown

• We know very little about the role of PDEs in P. falciparum biology and pathogenesis– Microarray data suggests the PDEs serve

different functions in different parasite life-cycles

• PDE inhibitors limit growth of P. falciparumin culture

Why Viagra Matters

• Due to drug industry’s interest in their role in ED, cardiovascular disease, and pulmonary diseases, there are libraries of PDE inhibitors available for screening against malaria– giving potential for “piggyback” development

of anti-malarials

PROBLEM:In order to screen compounds against malaria, we

need large quantities of soluble and catalytically active protein, which requires heterologous expression in E. Coli.

UNFORTUNATELY:Using conventional cloning techniques, P.

falciparum PDE constructs are able to be expressed but are insoluble and thus catalytically inactive

GOAL:

• To test new strategies to make soluble proteins of malaria PDEs

• SO THAT we can perform structural and biochemical studies on malaria PDEs

• SO THAT we can understand the role of PDE’s in malaria

• SO THAT we can develop new medications for malaria

Bioinformatics: FoldindexTM online tool

1. Design PCR primers and amplify PDE catalytic domains ORF from cDNA library

2. BP reaction: Clone PCR product into donor vector (pDONR), generating entry clone

Purify PCR products

3. LR reaction: Transfer ORF into destination vector (pNGWA), generating expression clone

GatewayTM

Clonase II Technology

Strategies for solubility

FoldindexTM

An online foldability prediction algorithm:• Predicts how much of a given protein sequence

is intrinsically folded • Rearranges Uversky algorithm, which uses the

Kyte & Doolittle prediction of hydrophobicity– positive values represent proteins (or domains) likely

to be folded– negative values represent those likely to be

intrinsically unfolded

Foldindex examples

• Folded protein:

• Unfolded protein:

Predicting solubility - Foldindex• Maximized "foldability" by cutting "unfoldable" regions,

truncating ORF while ensuring containment of catalytic domain

Catalytic Domain

Edited to maximize foldExample: Construct PDE0672

Unedited




Purify PCR products


GatewayTM



Gateway technology

• Described by Hartley et al– enables rapid cloning of one or more genes

into virtually any expression vector using site-specific and conservative recombination

– eliminates use of restriction enzymes and ligase

• Here, used to clone PDE construct into E. coli expression vector




Purify PCR products


GatewayTM


*clone PDE construct into E. coli expression

vector


“BP” reaction

2. BP reaction: Clone PCR product (attB) into donor vector (attP), generating entry clone (attL)

Used Foldindex to maximize chances of being “foldable”

PDE PDE

“BP” reaction – confirmed!

• Entry clone made with BP reaction- confirm with

diagnostic cut with restriction endonuclease (EcoRV), looking for shift in size

“BP” reaction

2. BP reaction: Clone PCR product (attB) into donor vector (attP), generating entry clone (attL)

Used Foldindex to maximize chances of being “foldable”

PDE PDE




Purify PCR products


GatewayTM



vector


“LR” reaction

3. LR reaction: Transfer GOI (attL) from entry clone into destination vector (attR), generating expression clone (attB)

How can we alter this to maximize our chances of making soluble protein?

PDE PDE

Optimizing destination vector for maximum solubility:

Maximizing “Solubility” with Nus-A fusion tag

Nus-A (N-utilizing substance A) is a solubility-promoting fusion tag

• Adapted as destination vector (pNGWA) for GatewayTM cloning technology

• Nus-A tagged expression vectors resulted in higher solubility when compared to 6-histidine tags

“LR” reaction


How can we alter this to maximize our chances of making soluble protein?

PDE PDE

“LR” reaction


PDE PDE

Nus-A

“LR” reaction – confirmed!

• Expression clonecreated– Verify using

diagnostic Xho1, Kpn1, digestion

“LR” reaction


PDE PDE

Nus-A

BigDye sequencing used to verify presence of PDE gene




Purify PCR products


GatewayTM



vector


Now that we have created an Expression Clone of PDE, It’s time to make protein!

• Transform expression clones (from LR reaction) into BL21star E. Coli, a good expression strain

• Manual induction with IPTG during log phase of bacterial growth

Making protein - results

• SDS-PAGE with Crude and Soluble (supernatant of centrifuged) protein– Predicted size 80-85 kDa

Soluble protein expressed for 2 of 4 PDE genes

Conclusions

• FoldindexTM algorithm facilitated solubility by predicting ORF regions with optimal foldability.

• Heterologous expression of two P. falciparumPDE genes was achieved in E. coli using an altered GatewayTM cloning expression system containing a Nus-A fusion protein.

• This is the first description of the use of Nus-A fusion for the heterologous expression of soluble P. falciparum PDE

Next steps

Next steps

• Soluble protein expressed will undergo purification by Nickel-MTA resin

• After purification, functional assays will be performed to determine catalytic activity

• If catalytic activity is present, protein will be used in X-ray crystallography for structural determination and biochemical assays for screening of PDE inhibitors lethal to malaria

• If catalytic activity not present, consider alternative Fusion tags with Gateway system

Acknowledgements

• Paul Pottinger (Professor extraordinaire)• Wes Van Voorhis • Van Voorhis / Buckner Lab

Purification of PCR products

• Needed due to potential attB primers and primer-dimers, which can:– recombine with the donor vector– increase background after transformation

• Invitrogen 30% PEG 8000 / 30mM MgCl2protocol

• Qiagen gel extraction protocol

Methodology – Manual induction

– Dilute 100 ul BL21star transformation into 4 ml LB w/Amp

– Incubate at 37oC until half-standard McFarlane

– Dilute 4 ml into 50 ml LB w/Amp & Carb– Incubate at 37oC until OD600 = 0.6-0.8 AU– Induced with 1mM IPTG– shook at 18oC for 4h & overnight

Methodology – Sonication & Extraction

– Resuspend pellet in 2 ml of lysis buffer (with imidazole, DTT, lysozyme, protease inhibitor)

– Sonicate bacterial suspension on ice, for 50 cycles

– Centrifuge 1ml of lysate at 13,000g for 10 min at 4°C, and transfer supernatant to a fresh tube (to be analyzed as soluble protein)

beyond viagra: novel use of nus-a fusion and gateway ... · gateway cloning technology in the...

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