High yield expression and efficient purification of deuterated human protein galectin-2

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dc.contributor.authorChen, XJen_AU
dc.contributor.authorWilde, KLen_AU
dc.contributor.authorWang, Hen_AU
dc.contributor.authorLake, Ven_AU
dc.contributor.authorHolden, PJen_AU
dc.contributor.authorMiddelberg, APJen_AU
dc.contributor.authorHe, LHen_AU
dc.contributor.authorDuff, APen_AU
dc.date.accessioned2014-03-20T04:36:13Zen_AU
dc.date.available2014-03-20T04:36:13Zen_AU
dc.date.issued2012-07-01en_AU
dc.date.statistics2014-03-20en_AU
dc.description.abstractStructural studies of biological macromolecules often require deuterated proteins, necessitating an effective bioprocessing strategy for high yield deuteration and purification. The fermentation and bioseparation studies reported here concern deuterated human protein galectin-2 mutant C57M (hGal-2), a protein showing potential for therapeutic applications. Using the vector pET-28a and a defined D2O based minimal medium with glycerol as the sole carbon source and kanamycin for selection, we have demonstrated that a high density of Escherichia coli expressing deuterated protein at a bench bioreactor scale (7L) can be achieved, with due attention to prevention of oxygen limitation. Yields achieved were 58 g\L biomass (wet weight) containing 0.7 g/L hGal-2. Affinity chromatography and ion-exchange chromatography were combined to achieve high purity as well as removal of hGal-2 aggregates, giving an overall yield of 1200 mg deuterated hGal-2. The deuterated hGal-2 was characterized and compared with the non-deuterated protein by size exclusion chromatography (SEC), HPLC, N-terminal sequencing, mass spectrometry (MS) and a dot blot immunoassay, showing that deuteration and subsequent purification did not impact the lactose binding and antibody recognition abilities of hGal-2. MS for both intact and trypsin-digested hGal-2 demonstrated that the extent of labeling of non-exchangeable hydrogen atoms by deuterium was (66 +/- 1)%, which provides sufficient contrast variation for structural studies using small angle neutron scattering. The fermentation and bioseparation method established in this work can be applied to process other deuterated proteins with high yield and purity, opening the way to advanced structural studies. © Institution of Chemical Engineers 2014.en_AU
dc.identifier.citationChen, X., Wilde, K. L., Wang, H., Lake, V., Holden, P. J., Middelberg, A. P. J., He, L., & Duff, A. P. (2012). High yield expression and efficient purification of deuterated human protein galectin-2. Food and Bioproducts Processing, 90(3), 563-572. doi:10.1016/j.fbp.2011.12.004en_AU
dc.identifier.govdoc4456en_AU
dc.identifier.issn0960-3085en_AU
dc.identifier.issue3en_AU
dc.identifier.journaltitleFood and Bioproducts Processingen_AU
dc.identifier.pagination563-572en_AU
dc.identifier.urihttp://dx.doi.org/10.1016/j.fbp.2011.12.004en_AU
dc.identifier.urihttp://apo.ansto.gov.au/dspace/handle/10238/5257en_AU
dc.identifier.volume90en_AU
dc.language.isoenen_AU
dc.publisherElsevieren_AU
dc.subjectAffinityen_AU
dc.subjectDeuterationen_AU
dc.subjectIon exchangeen_AU
dc.subjectChromatographyen_AU
dc.subjectProteinsen_AU
dc.subjectMass spectroscopyen_AU
dc.titleHigh yield expression and efficient purification of deuterated human protein galectin-2en_AU
dc.typeJournal Articleen_AU
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