Abstract
Chemical modification of protein is an important tool for studying protein structure and function. To avoid the loss of protein activity, site-specific protein modification has been extensively studied in last decades. The studies of this thesis focused on the developments of site-specific protein modification and immobilization of modified protein on solid support by fluorous- or DNA-tagged protein. The unique affinity interaction between fluorous molecules has been applied in many fields. We took advantage of the resistance of non-specific interaction by fluorous surface on protein microarray fabrication. Two strategies for site-specific modification of proteins with a fluorous tag were developed in this thesis. First, the target protein, enhance green fluorescent protein (eGFP), maltose binding protein (MBP), and glutathione transferase (GST), were expressed by intein expression system and their C-terminus were conjugated with cysteic acid contained fluorous tag by native chemical ligation (NCL). Second, the anti-RAC antibody was labeled with boronic acid contained fluorous tag through boronic acid-diol interaction. The fluorinated protein were site-specifically immobilized on fluorous solid support by simply mixing the fluorinated protein and solid support. The non-covalent fluorous-fluorous interaction were stable enough to withstand continuous washing and presented excellent performance to suppress the non-specfic adsorption. DNA biosensor technologies are currently under intense investigation owing to their great promise for rapid and low-cost detection of specific DNA sequence. In this thesis, the specific interaction between DNA base pairs was applied on the protein immobilization on the magnetic nanoparticles to investigation of the activity difference between free and immobilized enzymes. The target enzyme, RmlA and GalK, were site specifically modified with DNA at their C-terminus using 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction to give Enzyme-DNA. Then, these enzymes were assembled on DNA@MNPs through the sequence-specific hybridization properties of DNA. The captured enzymes were released from DNA@MNPs when the incubation temperature was higher than Tm of dsDNA. The results showed that the activity of RmlA-DNA is higher than those of RmlA-DNA-DNA@MNP and directly immobilized RmlA@MNP. However, the activity of GalK is identical as those of GalK-DNA-DNA@MNP and directly immobilized GalK@MNP. The enzyme-DNA was easily recovered by incubation with DNA@MNP and can be re-used after released from MNP by heating.