Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies

Romier, C.; Ben Jelloul, M.; Albeck, S.; Buchwald, G.; Busso, D.; Celie, P.H.N.; Christodoulou, E.; De Marco, V.; van Gerwen, S.; Knipscheer, P.; Lebbink, J.H.; Notenboom, V.; Poterszman, A.; Rochel, N.; Cohen, S.X.; Unger, T.; Sussman, J.L.; Moras, D.; Sixma, T.K.; Perrakis, A.

doi:10.1107/S0907444906031003

Acta Crystallographica Section D
Acta Crystallographica
Section D BIOLOGICAL CRYSTALLOGRAPHY

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Figure 1
(a) The suggested workflow for using the pET-MCN (pET multi-cloning and expression) system. See §

2.3 for additional details. (b) Co-expression of NFYB–NFYC heterodimer and NFYA–NFYB–NFYC heterotrimer from human transcription factor NFY (all subunits were truncated to their evolutionary conserved regions). All samples represent complexes retained on cobalt beads. MW, molecular-weight markers. Lanes 1–3, co-expression of His-tagged NFYC and non-tagged NFYB using the original pET15b/pACYC11b vectors (1) and the pET-MCN vectors either from two vectors (2) or a single vector with both genes on the promoter (3). Lane 4, same as lane 3 (bi-cistronic) but with no tag encoded in front of the NFYC coding gene (no retention). Lane 5, expression of His-tagged NFYA from pET-MCN vector. Lane 6, combination of the vectors used for lanes 4 and 5 (His-tagged NFYA and bi-cistronic non-tagged NFYB/NFYC complex) to produce the full complex; all three proteins are retained on the beads.

BIOLOGICAL
CRYSTALLOGRAPHY

ISSN: 1399-0047

Volume 62| Part 10| September 2006| Pages 1232-1242

doi:10.1107/S0907444906031003

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