Preparation of affinity membranes using thermally induced phase separation for one-step purification of recombinant proteins.

We synthesized several surfactant-like ligands and prepared affinity membranes by introducing them into porous polymeric membranes using the thermally induced phase separation method. The ligands (nitrilotriacetate, iminodiacetate, and glutathione) were successfully displayed on the surfaces of cellulose diacetate membranes. Membranes functionalized with nitrilotriacetate and glutathione captured and released hexahistidine-tagged enhanced green fluorescent protein (His-tag GFP) and glutathione S-transferase (GST) selectively under appropriate conditions.

Programmable protein-protein conjugation via DNA-based self-assembly.

Protein molecules were precisely arrayed on a designable DNA scaffold close to each other using a DNA aptamer. By adding a chemical cross-linker, the neighboring protein molecules were effectively and covalently cross-linked to each other without losing their activities.

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry.

We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K(d) was approximately 10⁻⁶ (M⁻¹) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme.

DNA-enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium.

We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA-enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein.

Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system.

We propose a novel method to prepare a DNA-protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni(2+). Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA-protein conjugate was formed and immobilized in the presence of Ni(2+) on the microplate.