Toshiaki Banzai

Properties of TiO2-polyacrylic acid dispersions with potential for molecular recognition.

Titanium dioxide (TiO2)/polyacrylic acid (PAA) (TiO2/PAA) particles were formed by mixing PAA and an acidic solution of TiO2 nanoparticles in dimethylformamide (DMF) followed by heat treatment. TEM and particle analysis showed that the resulting particles had a narrow size distribution. The colloid was very stable and aggregation was not observed over a wide pH range (3-9) or at high salt concentration. The residual carboxylic acid of PAA could be modified via EDC/NHS activation to form an amide bond with a protein. An antibody was attached to the hybrid nanoparticle and specific binding to antigen was monitored by surface plasmon resonance. The results suggest that TiO2/PAA nanoparticles are candidates as the base component of a photocatalytic system with potential for substrate selectivity.

Characterization of the Erythropoietin (EPO) Protein Production in the Recombinant Human Cultured Cells, in which the Exogenous EPO Gene was Introduced into Hypoxanthine Phosphoribosyl Transferase (HPRT) Gene Locus

The fields of biological industry need to develop stable and efficient protein production systems. We previously reported that green fluorescent protein (GFP) gene, integrated into hypoxanthine phosphoribosyl transferase (HPRT) gene locus, was expressed at the constant level throughout long-term cultivation without any drug selection. In this study, we developed and characterized recombinant human erythropoietin (EPO) producer cell lines. Three kinds of hprt gene-targeting vectors with a different EPO expression cassette were constructed. The one kind of the vector, phprt-IVS-GT-EPO #52 (#52), has both EPO cDNA sequence and intron in the expression cassette. The #52 gene-targeting vector was introduced into HT1080 cell by electroporation, and both gene-targeted and randomly-integrated clones were selected by G418/6-thioguanine (6TG) or G418 drug screening, respectively. Three gene-targeted clones and four randomly-integrated clones were picked up arbitrarily and evaluated for specific EPO production rates. Average and standard deviation values of three gene-targeted clones were 0.32 ± 0.05 pg cell–1 day–1 (CV = 14.6%), and the values of four randomly-integrated clones were 0.38 ± 0.22 pg cell–1 day–1 (CV = 59.1%). The low CV value of gene-targeted clones was also observed in the other two kinds of the targeting-vector (CV = 15.2% and 16.1%). The results show that the producer cell lines developed by gene-targeting into hprt locus are uniformity with respect to the specific production rates of a recombinant protein. This character could make it easier to select producer cell lines among many candidate cell lines with great diversity of production level. The gene-targeted clones maintained the specific EPO production rates during long-term cultivation, at least 60 days, without any selection drugs (G418 and/or 6TG). While the specific EPO production rates decreased to 50–60% of the initial values during 60 days cultivation without any drug selection in the randomly-integrated clones. The results show that the long-term stability of recombinant protein production rates achieved by the targeted integration into hprt locus is also applicable to the secreted EPO protein.