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

Chen, Xiaojing, Wilde, Karyn L., Wang, Hui, Lake, Vanessa, Holden, Peter J., Middelberg, Anton P. J., He, Lizhong and Duff, Anthony 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.2011.12.004


Author Chen, Xiaojing
Wilde, Karyn L.
Wang, Hui
Lake, Vanessa
Holden, Peter J.
Middelberg, Anton P. J.
He, Lizhong
Duff, Anthony P.
Title High yield expression and efficient purification of deuterated human protein galectin-2
Journal name Food and Bioproducts Processing   Check publisher's open access policy
ISSN 0960-3085
1744-3571
Publication date 2012-07-01
Sub-type Article (original research)
DOI 10.1016/j.fbp.2011.12.004
Volume 90
Issue 3
Start page 563
End page 572
Total pages 10
Place of publication London, United Kingdom
Publisher Elsevier
Language eng
Formatted abstract
Structural 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 (7 L) 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.
Keyword Affinity chromatography
Deuteration
Human galectin-2
Ion-exchange chromatography
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
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