Oriented Attachment of Recombinant Proteins to Agarose-Coated Magnetic Nanoparticles by Means of a β-Trefoil Lectin Domain

Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currently represents an active field of research. With this in mind, we have prepared and characterized agarose-coated maghemite nanoparticles to set up a platform for the attachment of recombinant proteins fused to the β-trefoil lectin domain LSL, a small protein that combines fusion tag properties with agarose-binding capacity. Analysis of the agarose-coated nanoparticles by dynamic light scattering, Fourier transform infrared spectroscopy, and thermogravimetric studies shows that decoupling particle formation from agarose coating provides better results in terms of coating efficiency and particle size distribution. LSL interacts with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes, which in turn can be dissociated ad hoc with the competing sugar lactose. Characterization of the complexes formed with the fusion proteins LSL-EGFP (LSL-tagged enhanced green fluorescent protein from Aquorea Victoria) and LSL-BTL2 (LSL-tagged lipase from Geobacillus thermocatenolatus) provided evidence supporting a topologically oriented binding of these molecules to the interface of the agarose-coated nanoparticles. This is consistent with the marked polarity of the β-trefoil structure where the sugar-binding sites and the N- and C-terminus ends are at opposed sides. In summary, LSL displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles.This work was partially supported by grant No. MAT2014-52069-R from the Spanish Ministry of Economy and Competitiveness (M.P.M.), by C-KBBE/3293 project from the European Commission (J.M.G.) and by grant BFU2010-17929 from the Spanish Ministry of Science and Innovation (J.M.M.). I.A. and Y.L. hold a predoctoral FPU fellowship from the Spanish Ministry of Economy and Competitiveness. A.G.R.-E. holds a predoctoral fellowship from a CSIC–CITMA collaborative project (ICMM, 2011-2014).Peer Reviewe

Directed, strong, and reversible immobilization of proteins tagged with a β-trefoil lectin domain: a simple method to immobilize biomolecules on plain agarose matrixes

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 (LSL 150), 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 LSL 150 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 LSL 150-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 LSL 150-agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL 150 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.Peer Reviewe

Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins

Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein¿support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.Peer Reviewe

Improving properties of a novel β-galactosidase from Lactobacillus plantarum by covalent immobilization

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license.-- This article belongs to the Section Natural Products.A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50°C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3′-galactosyl lactulose, has been described.This work has been sponsored by Consolider INGENIO 2010 CSD2007-00063 FUNC-FOOD(CICYT), the Spanish Ministry of Science and Innovation (Project CTQ2009-07568), CSIC(Intramural Project 200980I133) and S2009/AGR-1469 (ALIBIRD) (CAM).We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to α-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70°C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25°C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45°C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.Comunidad de MadridMinisterio de Educación y CienciaFundación Ramón ArecesDepto. de Ingeniería Química y de MaterialesFac. de Ciencias QuímicasTRUEpu