Plataformes científiques i Serveis
Expressió de proteïnes
Introducció
The IRB Barcelona Protein Expression Core Facility (PECF) provides a range of 'High Through-Put' (HTP) cloning and expression screening services to the local academic scientific community. The ability of HTP methods to clone and expression screen many constructs in parallel is an invaluable tool that can provide solutions to problems relating to protein expression or simply be used to rapidly expand the number of useful reagents (expression vectors) available to a research project. The facility now offers the following services:
- Custom HTP Cloning to generate Expression vectors. The facility will be capable of generating a microtitre plate of 96 expression-ready clones within 1-2 weeks of receiving template and primers. The plate may be comprised of either:
constructs of many different ‘target’ proteins from a single organism under investigation (i.e. a ‘Structural Genomics’ approach).
or, alternatively, multiple constructs of fewer, more complex proteins, where expression may have previously proven difficult and the investigator wishes to explore more expression options.
Constructs may contain defined N or C-terminal deletions of the proteins in combination with a choice of ‘fusion’ proteins (e.g. Maltose Binding Protein, MBP, Glutathione-S-Transferase, GST, or Small Ubiquitin-like MOdifier, SUMO) to aid the solubility and yield of the proteins of interest. The In-Fusion™ cloning technique and the pOPIN suite of vectors 1 will be used to generate expression constructs. A His6 tag is included in all constructs to simplify parallel expression screening and protein purification. - Expression Screening in E.coli. A microtitre plate of 96 (PEU or user-derived) expression clones may be screened in E.coli in approximately 1 week. The screen generally consists of the use of 2 expression strains, with expression in each strain being tested using both IPTG and Auto-Induction methods. Additional (DE3) E.coli strains may be incorporated into the screening process if required.
- Expression Screening in Mammalian, e.g. HEK293, cells-a microtitre plate of 96 (PEU or user-derived) expression clones may be screened in HEK293 cells in 1-2 weeks. The anticipated introduction of this process is during the third quarter of 2008.
Other services to be introduced during 2008 and 2009 include: recombinant baculovirus generation, expression screening in Sf9 cells, custom vector production, vectors for expression screening in P. pastoris or K. lactis.
Please contact Nick.berrow
irbbarcelona.org for more information on this facility.
1-the pOPIN vector suite was originally developed at the Oxford Protein Production Facility, see also:
Berrow, N.S., D. Alderton, S. Sainsbury, J. Nettleship, R. Assenberg, N. Rahman, D.I. Stuart, and R.J. Owens. 2007. A versatile ligation-independent cloning method suitable for high-throughput expression screening ap¬plications. Nucleic Acids Res. 35:e45.
In-Fusion™ is a trademark of Clontech Laboratories, Inc.
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