DEVELOPMENT OF A HIGH-THROUGHPUT

Transcription

1 DEVELOPMENT OF A HIGH-THROUGHPUT PROTEIN EXPRESSION SYSTEM USING BACMAM VIRUSES Pravin Mahajan SGC, Nuffield Department of Clinical Medicine University of Oxford SGC Toronto SGC Oxford

2 BACMAM (BACULOVIRUS MAMMALIAN) APPLICATIONS Cell Based Assays Tissue Engineering Protein Production Cancer Therapy BacMam Virus Production Vaccine VLP Production RNA Interference Protein Display

3 ADVANTAGES OF BACMAM EXPRESSION SYSTEM The virus is incapable of replicating in mammalian cells- BSL1 Baculovirus is not lytic in mammalian cells Baculovirus promoters are not active in mammalian cells Huge cloning capacity (at least 38 kb) Simultaneous expression of multiple genes No cytopathic effects No transfection reagent required High titres: ~1 x 10 8 pfu/ml Virtually endotoxin free

4 EXPRESSION SCREENING IN 24-WELL PLATES AND SCALE-UP, ADHERENT CULTURE- HEK293 Western blot on whole cell lysates But not all the constructs identified as +ve from the Western blot were scalable back to developing the screening method

5 ADHERENT CULTURE- MINIMAL SCALE FOR DETECTION BY COOMASSIE SDS-PAGE Transduced for 2 h, expressed for 72 h, IMAC-purified Cell number is limited by surface area

6 IDENTIFYING THE BOTTLENECKS Current expression screening methods use immunodetection techniques 1. Limitation on number of host cells that can be accommodated in adherent culture in smaller multi-well plates Action: suspension cell culture and medium that supports high density growth 2. Virus generated in insect cells, when added to the mammalian cells, causes severe abnormalities in size and shape Action: concentrate virus or media exchange 3. Transduction efficiency Action: vector engineering

7 VIRUS CONCENTRATION TO IMPROVE GENE DELIVERY CD 293 FreeStyle293 Pro293s ZMPSTE24 Sample Total protein yield from 15 ml (µg) FreeStyle 1: 40,000 x g : 80,000 x g- sucrose : 80,000 x g : PEG10, : PEG Pro293s-CDM

8 HDAC INHIBITOR, CELL LINE AND MEDIA TESTING- No inhibitor +Na-Butyrate +VPA 3ML SCALE Expi293F in FreeStyle293 medium Expi293F in Expi293 medium Expi293F cells can be used in Freestyle medium (2X cheaper) to clearly detect IMAC-purified protein by SDS-PAGE

9 BACMAM VECTOR ENGINEERING SacB SacB Bifunctional BacMam vector -insect and mammalian cells SacB

10 ADAPTATION OF PHTBV1.1 FOR LIC CLONING phtbv1.1 SacB

11 VECTOR COMPARISON RESULTS 1. pfb-mam-ct10hf-lic 2. pbacmam2-lic 3. pbacmam-diex-lic 4. pbacmam2-diex-lic 5. phtbv1.1-lic* phtbv1.1-lic has given higher protein yield Bifunctional vectors have produced proteins in insect and human cells

12 Transient Transfection TRANSDUCTION EFFICIENCY Na Butyrate Transduction, 5% virus Transduction, 10% virus Na Butyrate Na Butyrate

13 HIGH-THROUGHPUT EXPRESSION SCREENING - 3 ML SCALE, 24-WELL BLOCKS * * * * * * * * * * * * * * * * * Intracellular Proteins * * * * * * * * * * Integral Membrane Proteins

14 WHY USE HIGHER EUKARYOTIC EXPRESSION SYSTEM } }? Parallel testing of multiple constructs of human proteins in E. coli. Soluble expression in >60% of targets. Targets that failed to express in E. coli were then tested in baculovirus vectors. 40% of the proteins were purified in sufficient amount and purity for crystallisation. Opher Gileadi

15 COMPARING DIFFERENT EXPRESSION SYSTEMS Target Soluble Protein Expression in IMP Expression in Target E. coli Insect Cells Mammalian Cells E. coli Insect Cells Mammalian Cells Sol-1 IMP-1 NA Sol-2 IMP-2 NA Sol-3 IMP-3 NA Sol-4 NT IMP-4 NA Sol-5 IMP-5 NA Sol-6 IMP-6 NA Sol-7 IMP-7 NA Sol-8 NT IMP-8 NA Sol-9 NT IMP-9 NA Sol-10 IMP-10 NA Sol-11 IMP-11 NA Sol-12 IMP-12 NA Sol-13 IMP-13 NA Sol-14 IMP-14 NA Sol-15 IMP-15 NA Sol-16 IMP-16 NA IMP-17 NA Key: IMP-18 NA expression IMP-19 NA no expression IMP-20 NA NA not applicable IMP-21 NA NT not tried

16 BACMAM EXPRESSION PROCESS 96-well format H12 = + control 4x24-well plates (96 h) 4x24-well blocks (48 h)

17 CONCLUSION First 3ml expression screening method for mammalian cells that allows assessment of protein bands on SDS-PAGE gels stained with Coomassie Blue (no immunodetection required) A measure of protein purified and not just protein expressed- better predictability of scalable constructs The methodology is successfully applied to large scale expressions

18 ACKNOWLEDGEMENTS Biotechnology Nicola Burgess-Brown PI Katherine Ellis Dong Wang Katarzyna Kupinska Shubhashish Mukhopadhyay Leela Shrestha Rod Chalk Claire Strain-Damerell Opher Gileadi Jon Elkins Alex Bullock Catrine Johansson Wyatt Yue Integral Membrane Protein Group Liz Carpenter PI Andrew Quigley Yin Dong Mariana Grieben FUNDING PARTNERS The Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, Takeda, the Ontario Ministry of Economic Development and Innovation, and the Wellcome Trust.