Results from an expression screen performed within the Protein Production Network Sweden (PPNS) Dr Malin Bäckström

Mammalian Protein Expression (MPE) core facility

P4EU meeting, Vienna, Dec 2014

Core Facilities at the Sahlgrenska Academy, the Medical Faculty of the University of Gothenburg

• EBM - Laboratory for Experimental Biomedicine • CPI - Centre for Physiology and BioImaging • MPE - Mammalian Protein Expression • Proteomics • CCI - Centre for Cellular Imaging • Genomics • Bioinformatics

http://www.cf.gu.se

PPNS: Protein Production Network in Sweden

Umeå University

Uppsala University Karolinska Institute, Stockholm

Linköping University Gothenburg University

Lund University

PPNS: Protein Production Network in Sweden

PEP, Umeå (E. coli)

Protein Science facility, Stockholm (E. coli) (Science for Life Lab, former SGC)

MPE, Göteborg (Mammalian cells) Swedish NMR centre (cell-free)

Astra Zeneca, Mölndal

LP3, Lund (E. coli, BEVS) Max IV

PPNS: Protein Production Network in Sweden

• First PPNS meeting in 2013 • Aim: to make protein production methods and competence

available to the Swedish academic life science community

• Common funding? • Common expression screen as a first step towards becoming

a distributed national infrastructure for protein production

PPNS: Time frame for an expression screen in 2014 • February: Decision to perform an expression screen • June: selection of proteins among nominations from all

centres involved • August: Vectors ready and distributed (PSF) • October: Experiments performed at the different platforms • November: Presentation of results at an internal meeting

in Gothenburg • December: Writing of common report • Spring 2015: Present report to funding bodies;

possibilities for grant application?

PPNS: Proteins selected for the expression screen

• Full-length and one functional domain constructs were included • C-terminal His- and flag-tags were used

Expression vectors were prepared at the Protein Science Facility in Stockholm by LIC cloning

• E. coli and cell-free: pNIC-CH2

• Mammalian cells: pcDNA3-CT10HF-LIC

• BEVS: 1) pFB-CT10HF-LIC (Bac-to-Bac) (failed) 2) pVL1392-CH2 (FlashBac)

E. coli expression at the Protein Science Facility (PSF), Karolinska Insitute, Stockholm

E. coli expression at the Protein Expression Platform (PEP) Umeå

Cell-free in vitro expression at the Swedish NMR centre (SNC), Gothenburg

Baculovirus expression at LP3, University of Lund (only full-length constructs)

1. eGFP 2. CA IX 3. PARP6 4. PRODH 5. YjbH 6. ZG16 7. TEV

Mammalian expression at MPE, University of Gothenburg, (I) CHO-S cells

1. CA IX FL 2. CA IX domain 3. GFP 4. PARP FL 5. PARP domain 6. PRODH domain 7. YjbH FL 8. YjbH domain 9. ZG16 FL 10. ZG16 11. TEV

Mammalian expression at MPE, University of Gothenburg, (II) 293F cells

1. CA IX FL 2. CA IX domain 3. GFP 4. PARP FL 5. PARP domain 6. PRODH domain 7. YjbH FL 8. YjbH domain 9. ZG16 FL 10. ZG16 11. TEV

Summary of results from PPNS screen

An important result is also that we have learnt what techniques are available at the different Swedish protein expression platforms, so that we can direct researchers to the different centres when we get inquiries about different proteins.

Continued work within PPNS

• Written report finalized in December 2014

• Distribution of report to Swedstruct, Swedish Science Council, Science for Life Laboratories…

• Potential application for funding for becoming a national resource within protein production during 2015