Directed, Strong, and Reversible Immobilization of Proteins Tagged with a β-Trefoil Lectin Domain: A Simple Method to Immobilize Biomolecules on Plain Agarose Matrixes

Abstract

A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus. Such a domain (LSL150), recently described as a novel fusion tag, is based on a β-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL150 to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the LSL150–agarose interaction. Furthermore, this latter interaction is temperature dependent since it is 4-fold stronger when the immobilization takes place at 25 °C than when it does at 4 °C. The strongest lectin–agarose interaction is also quite stable under a survey of different conditions such as high temperatures (up to 60 °C) or high organic solvent concentrations (up to 60% of acetonitrile). Notably, the use of cross-linked agarose would endow the system with more robustness due to its better mechanical properties compared to the noncross-linked one. The stability of the LSL150–agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL150 lectin domain exhibits interesting structural features as an immobilization domain that makes it suitable to reversibly immobilize industrially relevant enzymes in very simple carriers as agarose.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…material, internal architecture), controlling the rate of enzyme adsorption was also important when considering homogeneous distribution of bound enzymes in porous supports [87][88][89]. Moreover, the degree of reversibility of the adsorption also affected the final distribution of enzyme within Table 3 the support [46,90]. It was also shown that degree of homogeneity of enzyme adsorption had a decisive influence on the specific activity of the heterogeneous biocatalyst [87,88] (Table 2).…”

“…Methods capable of revealing the conformation of proteins in solution and on solid surface would therefore be required to test and eventually establish correlations between the degree of conformational distortion and the residual specific activity of the immobilized enzyme. In a similar manner, stability of immobilized enzymes could be analyzed where the degree of [90] stability enhancement might be related to detectable and ideally also quantifiable conformational distortions in the immobilized enzyme [19,21,91]. Problem is that only few of the various spectroscopic techniques applied routinely to the study of protein conformations in solution are suitable for analysis of proteins on surface, especially that of a porous solid support.…”

Single‐Particle Studies to Advance the Characterization of Heterogeneous Biocatalysts

“…On the other hand, the single-particles tudies of green fluorescent protein tagged with al ectin domain allowed monitoring the spatial distribution of the immobilized proteins over the time. [37] This study revealed that protein is primarily immobilized on the outer surfaceo fp orous agarose beads, but then gradually colonizes the whole microstructure of the agarose particle( 50-150 mm) along 30 minutes.T hese results evidence that the interactions between the agarose surface and the lectin domain are dynamic and suggest an intraparticle association/dissociation equilibrium between the lectin and the sugars forming the agarose fibers. Such equilibrium seems to enable the reorganization of the spatial distribution inside the particles upon the immobilization.…”

“…The immobilization rate can be easily modulated by controlling the immobilization chemistry (nature and density of the reactive groups) and the immobilization conditions (presence of competitors, pH, temperature). On the other hand, the single‐particle studies of green fluorescent protein tagged with a lectin domain allowed monitoring the spatial distribution of the immobilized proteins over the time . This study revealed that protein is primarily immobilized on the outer surface of porous agarose beads, but then gradually colonizes the whole microstructure of the agarose particle (50–150 μm) along 30 minutes.…”

A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

“…Although this galactose binding domain has two galactose binding sites ( Figs 1 and 2 ), the binding affinities are relatively weak. It has been shown that the galactose binding domain from the mushroom hemolytic toxin LSLa binds to agarose beads in a dynamic manner 36 . The rapid dissociation of this domain from the beads and its rebinding leads to a fast infiltration of the galactose binding domain from the bead surface to the centre.…”