Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.
We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain ; Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
Daniel
D
Bello-Gil
Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
Beatriz
B
Maestro
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain.
Jennifer
J
Fonseca
Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
Juan M
JM
Feliu
Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
Víctor
V
Climent
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain.
Jesús M
JM
Sanz
D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006108:Graphite; D055767:Immobilized Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D006108:Graphite; D000581:Amidohydrolases; C019665:amidase