The lectin Cbol was purified to apparent homogeneity by affinity chromatography on Sephadex G-50, in which Cbol was quantitatively retained in the cross-linked dextran gel column and was desorbed with d
-glucose, providing strong evidence of its carbohydrate-binding properties (Fig. 1
). This procedure has widely been used for the purification of Diocleinae lectins (Cavada et al.
). About 80 mg purified lectin was obtained from 1 g powdered seeds. The purified protein showed haemagglutination activity towards native and enzyme-treated rabbit erythrocytes. SDS–PAGE confirmed the purity of the lectin and its similarity to other Diocleinae lectins, showing a main α chain and β and γ fragments (Fig. 1
). These results suggest that Cbol is a ConA-like lectin and undergoes the same post-translational process of circular permutation as first described by Carrington et al.
). This process involves proteolytic cleavage of the precursor at an internal site, resulting in the γ and β fragments. These fragments are religated in an inverse DNA-coded order, producing the active α chain (α = β + γ; Carrington et al.
; Chrispeels et al.
; Min & Jones, 1994
). The β and γ fragments observed in the SDS–PAGE (Fig. 1
) are non-religated products of this process and the α chain is the mature protein. The Diocleinae lectins used for comparison, ConBr and CGL, have previously been characterized and their three-dimensional structures have been determined (PDB code 1azd
, Sanz-Aparicio et al.
; PDB code 2d7f
, Delatorre et al.
Figure 1 Affinity chromatography and SDS–PAGE of the purified C. boliviana lectin (Cbol). (a) Sephadex G-50 chromatogram; the bonded protein was eluted with 0.1 M
d-glucose. (b) Lane 1, SDS–PAGE showing protein (more ...)
Crystals were obtained after a week using condition Nos. 30, 31, 34 and 38 of Hampton Crystal Screen I. Condition No. 34 was chosen to be optimized; it contained 0.1 M
sodium acetate trihydrate pH 4.6 and 2.0 M
sodium formate. To optimize the crystallization conditions, the concentration of sodium formate was modified from 1 to 6 M
and buffers of differing pH were tested. Crystals suitable for diffraction experiments (Fig. 2) were obtained under the condition 0.1 M
HEPES pH 7.5 and 3.0 M
sodium formate. The crystals obtained provided a data set extending to 1.5 Å resolution, which was scaled in the resolution range 46.56–1.60 Å. The C. boliviana
lectin crystal belongs to the centred monoclinic space group C
2, with unit-cell parameters a
= 126.7, b
= 66.6, c
= 64.9 Å, α = 90.0, β = 120.8, γ = 90.0°. The calculated value of the Matthews coefficient (Matthews, 1968
), based on the molecular weight of 25.5 kDa, indicated a solvent content of 46.7%, which corresponds to the presence of a dimer in the asymmetric unit. Data-collection statistics are shown in Table 1. The preliminary crystal structure of Cbol was determined by standard molecular-replacement methods using the program MOLREP
(Vagin & Teplyakov, 1997
). Various monomers were tested for molecular replacement and the best result was obtained using CGL (PDB code 2d7f
; Delatorre et al.
) as a structural model. The best solution had a final correlation coefficient of 0.705 and an R
factor of 0.424. After placing the molecule in the unit cell, rigid-body refinement was performed using the REFMAC
5 program. Refinement resulted in a model with an R
of 0.363 and an R
factor of 0.359. Primary sequence determination by mass spectrometry and Edman degradation as well as solution of the crystal structure are in progress.
Crystals of C. boliviana lectin.
Statistics of data collection