Improving the Tractability of Membrane Protein ...

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reproducibly produce high yielding and structurally stable recombinant GPCRs, ... (B) Illustrates the strategy for designing the construct with a GFP tag by ligating (A) into ... Contact: Zharain Bawa. Email: [email protected]. 5'. 3'. 10x. His. Tag ... Tonge S.R. and Tighe B.J. (2001) Responsive hydrophobicallly associating ...
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Zharain Bawa1, Markus Ganzlin2 and Roslyn M. Bill1

of Life and Health Sciences, Aston University, Birmingham B4 7ET 2AstraZeneca Ltd., Alderley Park, Macclesfield, Cheshire, SK10 4TF (Sponsor of BBSRC CASE Award)

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Contact: Zharain Bawa Email: [email protected]

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Improving the Tractability of Membrane Protein Production in Yeast

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yTHCBMS1 strain 18.11

TM6* strain 4.59

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Mean (n=3)

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Specific Binding (fmols /mg protein)

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(B) Specific Binding of hA2aR after SMA solubilisation

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Figure 4. (A) Clone 26 Immunoblot Result

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4. André et al (2006) Enhancing functional production of G-protein coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen Protein Sci. 15:1115-1126

5. Lundstrom et al (2006) Structural genomics on membrane proteins: comparison of more than 100 GPCRs in three expression systems J. Struct. Funct. Genomics 7: 77-91 6. Knowles et al (2009) Membrane proteins solubilised intact in lipid containing nanoparticles bounded by styrene maleic acid coppolymer J. Am. Chem. Soc. 131: 7484-7485 7. Tonge S.R. and Tighe B.J. (2001) Responsive hydrophobicallly associating polymers: a review of structure and properties Adv. Drug Delivery Reviews 53: 109-122 8. Drew et al (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification Protein Sci. 14: 2011-2017

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2. Bonander et al (2009) Altering the ribosomal subunit ratio in yeast maximises recombinant protein yield Microbial Cell Factories 8:10

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References 1. Holmes et al (2009) Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime Microbial Cell Factories 8:35

3. Otterstedt et al (2004) Switching the mode of metabolism in the yeast Saccharomyces cerevisiae EMBO Reports 5:532-537

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Conclusions & Future Perspectives Objective I The preliminary DoE results suggest no effect from the screening conditions on OD595 and total protein yields. However, radio-ligand binding experiments confirmed activity of the hA2aR expressed in the three S. cerevisiae strains subjected to the DoE screening conditions. Furthermore, binding activity was increased significantly when subjected to specific DoE input conditions in a strain-dependent manner. These growth conditions will be applied to 3L Bioreactor cultivations of all three S. cerevisiae strains expressing hA2aR to generate large amounts of correctly-folded receptor for further downstream studies. Objective II Optimal conditions retrieved from Objective I, will be used to scale-up GPCR production in 3 L Bioreactors. Feeding strategies will be explored in more detail for both P. pastoris and S. cerevisiae species for optimal protein production. Objective III Studies on solubilisation with SMA will continue by investigating variables associated with the polymer.

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Figure 5. Specific binding after thermostability tests for SMA and DDM solubilised hA2aR

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• A different clone, clone 27 was also solubilised with SMA and compared to dodecyl maltoside (DDM) detergent solubilisation. The solubilised material from both methods were subjected to thermostability studies and specific binding performed (Figure 5). Specific binding was found to be equivalent for both DDM and SMA solubilised material and furthermore, the SMA solubilised material showed more stable receptors after 50 C heat treatment.

Figure 2. SMA in the presence of membrane (lipid bilayer) and protein. • Specific radio-ligand binding assays carried out on solubilised material

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Lane 1: 250 kDa ladder

Solubilised membrane protein and lipid bilayer

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Mean specific radio-ligand binding activity with 3[H]ZM241385 antagonist (fmols/mg protein; n=3)

Objective II and III – Scale Up and Purification Results • The Bioreactor (3L) cultivation and purification of clone 26 P. pastoris with hA2aR yielded good total protein (2.8 g) and immunoblot signal (Figure 4A). This clone was SMA solubilised and specific binding performed (Figure 4B).

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Objective III – Purification Methods • P. pastoris hA2aR membranes solubilised with Styrene Maleic Acid (SMA)6,7 as opposed to traditional detergent solubilisation (Figure 2).

Styrene Maleic Acid (SMA)

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DoE Input Conditions

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Ammonium hydroxide pressure vessel

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Figure 1. Micro-24 Bioreactor System and single-use cassette holding 24 independently controlled Bioreactors.

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Table 2. DoE screening conditions yielding significantly different binding activities of hA2aR expressed in S.cerevisiae wild type, yTHCBMS1 and TM6* strains

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• Initial DoE experiments indicated that there was no significant effect of the screening conditions on OD595 (5.0-6.0) and total protein yield (130-250 µg). However, radio-ligand binding results showed significant differences in the amount of correctly folded receptor from the different screening conditions for all three S. cerevisiae strains (Table 2).

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Enterokinase Cleavage Site

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Figure 3. (A) Construct shows hA2aR along with specific tags and cleavage sites (enterokinase cleavage site within the FLAG tag sequence). (B) Illustrates the strategy for designing the construct with a GFP tag by ligating (A) into an existing pPICZαA – GFPUV vector.

Objective II – Scale Up Methods • Methylotrophic yeast, Pichia pastoris wild type strain integrated with hA2aR cultured in 3L Bioreactor at standard conditions • Total protein yield, immunoblot analysis and radio-ligand binding assays investigated

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Optical Density (OD595), total protein yield and radio-ligand binding assays were considered as “read-outs” of the DoE.

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Objective I – Screening Results • Designed two versions of hA2aR constructs, one with Green Fluorescent Protein (GFP)8 and one without. This will enable a quick screening method for positive GPCR expression for all the DoE experiments (Figure 3A and 3B). It will also facilitate localisation studies for the GFP-containing constructs.

Table 1. DoE input conditions of temperature, pH, dO and dimethyl sulphoxide (DMSO)4,5 presence/absence.

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• Explore novel GPCR purification methods for downstream structural analysis

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• Evaluate yeast cultivation regimes for large scale production

Aligns with Drug Discovery Pipeline

Objective I - Screening Methods • Designed novel expression construct for Human Adenosine 2a Receptor (hA2aR) incorporating detection and purification tags and cleavage sites • Expression experiments were screened via statistical “Design of Experiments” (DoE) approach1 (Table 1) and performed on Micro24 Bioreactor (PALL Corporation, Figure 1). DoE’s are an effective way of investigating the impact of multiple conditions whilst reducing the overall number of required experiments. Saccharomyces cerevisiae wild type strain, yTHCBMS1 over expression strain2 and TM6* respiratory strain3 were used in these experiments.



Objective III Purification

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• Construct designs for GPCR expression in yeast • Screen optimal growth conditions for yeast

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Objective II Scale Up

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Introduction Over 50% of clinically marketed drugs target membrane proteins, in particular G Protein-Coupled Receptors (GPCRs). One of the biggest challenges pharmaceutical industries face is to reproducibly produce high yielding and structurally stable recombinant GPCRs, integral to drug design. This project addresses three main objectives which are to develop efficient ‘smartthroughput’ screening methods for recombinant GPCR production in yeast; to upscale optimised cultivation regimes for GPCRs and to investigate recombinant GPCR isolation techniques from yeast cells to enable downstream structural studies.