Beyond Viagra: Novel use of Nus-A fusion and Gateway cloning technology in the heterologous expression of Plasmodium falciparum phosphodiesterases

Transcription

1 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

2 Malaria Caused by infection by Plasmodium species 300 million cases per year Directly causes 1 million deaths per year $12 Billion lost revenue in Africa

3 We need better anti-malarials! Increasing drug resistance Poor efficacy of current medications Does the answer lie in inhibitors of phosphodiesterases (PDE)?

4 Where have I heard of Phosphodiesterases (PDE)? Enzymes involved in intra- and intercellular signaling A B Adenylyl Cyclase Guanylyl Cyclase G AMP PDE ATP camp cgmp PDE GTP GMP cascade of biochemical reactions (Ca ++ dependent PKA, others)

5 No, really Where else have I heard of Phosphodiesterases (PDE)?

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7 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 lifecycles PDE inhibitors limit growth of P. falciparum in culture

8 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

9 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

10 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

11 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

12 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

13 Foldindex TM 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

14 Foldindex examples Folded protein: Unfolded protein:

15 Predicting solubility - Foldindex Maximized "foldability" by cutting "unfoldable" regions, truncating ORF while ensuring containment of catalytic domain Example: Construct PDE0672 Unedited Edited to maximize fold Catalytic Domain

16 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

17 Gateway technology Described by Hartley et al enables rapid cloning of one or more genes into virtually any expression vector using sitespecific and conservative recombination eliminates use of restriction enzymes and ligase Here, used to clone PDE construct into E. coli expression vector

18 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology *clone PDE construct into E. coli expression vector Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

19 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology *clone PDE construct into E. coli expression vector Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

20 BP reaction 2. BP reaction: Clone PCR product (attb) into donor vector (attp), generating entry clone (attl) PDE PDE Used Foldindex to maximize chances of being foldable

21 BP reaction confirmed! Entry clone made with BP reaction - confirm with diagnostic cut with restriction endonuclease (EcoRV), looking for shift in size

22 BP reaction 2. BP reaction: Clone PCR product (attb) into donor vector (attp), generating entry clone (attl) PDE PDE Used Foldindex to maximize chances of being foldable

23 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology *clone PDE construct into E. coli expression vector Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

24 LR reaction 3. LR reaction: Transfer GOI (attl) from entry clone into destination vector (attr), generating expression clone (attb) PDE PDE How can we alter this to maximize our chances of making soluble protein?

25 Optimizing destination vector for maximum solubility:

26 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 Gateway TM cloning technology Nus-A tagged expression vectors resulted in higher solubility when compared to 6- histidine tags

27 LR reaction 3. LR reaction: Transfer GOI (attl) from entry clone into destination vector (attr), generating expression clone (attb) PDE PDE How can we alter this to maximize our chances of making soluble protein?

28 LR reaction 3. LR reaction: Transfer GOI (attl) from entry clone into destination vector (attr), generating expression clone (attb) PDE PDE Nus-A

29 LR reaction confirmed! Expression clone created Verify using diagnostic Xho1, Kpn1, digestion

30 LR reaction 3. LR reaction: Transfer GOI (attl) from entry clone into destination vector (attr), generating expression clone (attb) PDE PDE Nus-A

31 BigDye sequencing used to verify presence of PDE gene

32 Strategies for solubility Bioinformatics: Foldindex TM online tool 1. Design PCR primers and amplify PDE catalytic domains ORF from cdna library Gateway TM Clonase II Technology *clone PDE construct into E. coli expression vector Purify PCR products 2. BP reaction: Clone PCR product into donor vector (pdonr), generating entry clone 3. LR reaction: Transfer ORF into destination vector (pngwa), generating expression clone

33 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

34 Making protein - results SDS-PAGE with Crude and Soluble (supernatant of centrifuged) protein Predicted size kda Soluble protein expressed for 2 of 4 PDE genes

35 Conclusions Foldindex TM algorithm facilitated solubility by predicting ORF regions with optimal foldability. Heterologous expression of two P. falciparum PDE 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

36 Next steps

37 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

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

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40 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 MgCl 2 protocol Qiagen gel extraction protocol

41 Methodology Manual induction Dilute 100 ul BL21star transformation into 4 ml LB w/amp Incubate at 37 o C until half-standard McFarlane Dilute 4 ml into 50 ml LB w/amp & Carb Incubate at 37 o C until OD 600 = AU Induced with 1mM IPTG shook at 18 o C for 4h & overnight

42 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)