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 LSL150, 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. LSL150interacts 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, LSL150displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles.
Departamento de Cristalografía y Biología Estructural, Instituto Rocasolano, CSIC , Serrano 119, 28006 Madrid, Spain.
Iván
I
Acebrón
Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de Materiales de Madrid, CSIC , Sor Juana Inés de la Cruz, 3, Cantoblanco, 28049 Madrid, Spain.
Amalia G
AG
Ruiz-Estrada
Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de Materiales de Madrid, CSIC , Sor Juana Inés de la Cruz, 3, Cantoblanco, 28049 Madrid, Spain.
Yurena
Y
Luengo
Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de Materiales de Madrid, CSIC , Sor Juana Inés de la Cruz, 3, Cantoblanco, 28049 Madrid, Spain.
María Del Puerto
MD
Morales
Departamento de Biocatálisis, Instituto de Catálisis, CSIC , Campus UAM, Cantoblanco, 28049 Madrid, Spain.
José Manuel
JM
Guisán
Departamento de Cristalografía y Biología Estructural, Instituto Rocasolano, CSIC , Serrano 119, 28006 Madrid, Spain.
José Miguel
JM
Mancheño
D005290:Ferric Compounds / Q000737:chemistry*; D000070116:Lotus / Q000737:chemistry*; D008958:Models, Molecular; D053758:Nanoparticles / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry*; D000072417:Protein Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry*
D005290:Ferric Compounds; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; C000499:ferric oxide; D012685:Sepharose