Glycoconjugate Journal

, Volume 27, Issue 4, pp 461–476 | Cite as

High-yield production, refolding and a molecular modelling of the catalytic module of (1,3)-β-d-glucan (curdlan) synthase from Agrobacterium sp.

  • Maria Hrmova
  • Bruce A. Stone
  • Geoffrey B. Fincher


Biosynthesis of the (1,3)-β-d-glucan (curdlan) in Agrobacterium sp., is believed to proceed by the repetitive addition of glucosyl residues from UDP-glucose by a membrane-embedded curdlan synthase (CrdS) [UDP-glucose: (1,3)-β-d-glucan 3-β-d-glucosyltransferase; EC]. The catalytic module of CrdS (cm-CrdS) was expressed in good yield from a cDNA encoding cm-CrdS cloned into the pET-32a(+) vector, containing a coding region for thioredoxin, and from the Champion™ pET SUMO system that possesses a coding region of a small ubiquitin-related modifier (SUMO) partner protein. The two DNA fusions, designated pET-32a_cm-CrdS and SUMO_cm-CrdS were expressed as chimeric proteins. High yields of inclusion bodies were produced in E. coli and these could be refolded to form soluble proteins, using a range of buffers and non-detergent sulfobetaines. A purification protocol was developed, which afforded a one-step on-column refolding and simultaneous purification of the recombinant 6xHis-tagged SUMO_cm-CrdS protein. The latter protein was digested by a specific protease to yield intact cm-CrdS in high yields. The refolded SUMO_cm-CrdS protein did not exhibit curdlan synthase activity, but showed a circular dischroism spectrum, which had an α/β-type-like conformation. Amino acid sequences of tryptic fragments of the SUMO_cm-CrdS fusion and free cm-CrdS proteins, determined by MALDI/TOF confirmed that the full-length proteins were synthesized by E. coli, and that no alterations in amino acid sequences occurred. A three-dimensional model of cm-CrdS predicted the juxtaposition of highly conserved aspartates D156, D208, D210 and D304, and the QRTRW motif, which are likely to play roles in donor and acceptor substrate binding and catalysis.


Escherichia coli Family GT2 transferases Heterologous expression Homology model Integral membrane proteins pET-32a(+) and Champion™ pET SUMO vectors 



Carbohydrate-active enzymes


N-cyclohexyl-2-aminoethanesulfonic acid


Circular dichroism


Catalytic module of CrdS


Curdlan synthase


Ethylenediaminetetraacetic acid


Extracellular and capsular polysaccharides


Glycoside transferase(s)


Hydrophobic cluster analysis


Immobilized metal affinity chromatography




Matrix-assisted laser desorption/ionization-time of flight


Mass spectrometry


Relative molecular mass


Non-detergent sulfobetaine


Protein data bank


Phenylmethylsulphonyl fluoride


Sodium dodecylsulfate


Small ubiquitin-related modifier




Uridine-diphosphate glucose





This work was supported by grants from the Australian Research Council (to GBF, BAS and MH). We are grateful to Dr Vilma Stanisich from La Trobe University (Australia) for providing pVS1512 plasmid and to Dr Christopher J. Bagley from the Adelaide Proteomics Centre (Australia) for mass spectrometry analyses.

Supplementary material

10719_2010_9291_Fig8_ESM.jpg (1.7 mb)
Fig. 1S

Plate layout of iFold Protein Refolding System 2 of 96 conditions ( used for refolding of chimeric cm-CrdS proteins (cf. Fig. S2). A negative control is in formulation 91. (JPEG 901 kb)

10719_2010_9291_MOESM1_ESM.eps (9.3 mb)
High Resolution Image (EPS 9557 kb)
10719_2010_9291_Fig9_ESM.jpg (902 kb)
Fig. 2S

Immuno-blot analyses of inclusion bodies of the chimeric cm-CrdS protein produced by the pET-32a_cm-CrdS and SUMO_cm-CrdS expression vectors and refolded in iFOLD protein Refolding System 2 (95 conditions and a negative control in formulation 91 is circled in red). Inclusion bodies of chimeric cm-CrdS refolded in individual conditions were applied to nitrocellulose filters and immuno-blot analyses were performed as described in Fig. 4. The individual refolding conditions are indicated by numbers on the left- and right-hand sides of the dot-blots. The highest protein yields, circled in green, were analysed by SDS-PAGE and the SDS-PAGE profiles are shown underneath each dot-blot. Approximately 10–15 µl of refolded proteins were applied in each site. (JPEG 1788 kb)

10719_2010_9291_MOESM2_ESM.eps (6.5 mb)
High Resolution Image (EPS 6668 kb)
10719_2010_9291_Fig10_ESM.jpg (276 kb)
Fig. 3S

Time-course of refolding of inclusion bodies of the chimeric cm-CrdS protein produced by the pET-32a_cm-CrdS and SUMO_cm-CrdS expression vectors in 25 mM CHES buffer, pH 8 with 0.5, 0.25, 0.125, 0.064, 0.032 and 0 M NDSB-256. A negative control (water) is shown. Protein refolding was monitored during the 0.3 h to 72 h time interval, after initiation of refolding by quantifying A340. The optimal refolding conditions, indicating concentrations of NDSB-256 and time intervals are indicated in each panel. (JPEG 275 kb)

10719_2010_9291_MOESM3_ESM.eps (327 kb)
High Resolution Image (EPS 327 kb)


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Maria Hrmova
    • 1
  • Bruce A. Stone
    • 2
  • Geoffrey B. Fincher
    • 1
  1. 1.Australian Centre for Plant Functional Genomics, School of Agriculture, Food and WineUniversity of AdelaideGlen OsmondAustralia
  2. 2.School of BiochemistryLa Trobe UniversityMelbourneAustralia

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