Optimization of production and characterization of a recombinant soluble human Cripto-1 protein inhibiting self-renewal of cancer stem cells.

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Both Pdx-1 and NeuroD1 genes are requisite for the maintenance of insulin gene expression in ES-derived differentiated cells.

Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. We have established an ES cell line in which exogenous Pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus and succeeded to produce insulin-producing cells. The Pdx-1 expressing final differentiated insulin-positive cells can be maintained for more than 2 months. However, in spite of their induced expression of Pdx-1, the repeated passages of cells lost their capacity to express insulin and NeuroD1 gene. Forced expression of NeuroD1 gene by adenoviral vector in these cells restored the expression of insulin. These results suggested that maintenance of the property of insulin-producing cells derived from ES cells could be achieved by synergistic expression of Pdx-1 and NeuroD1.

Analyses of microbial desulfurization reaction of alkylated dibenzothiophenes dissolved in oil phase.

The kinetics of the oil/water two-phase reaction system was analyzed, and the reaction was carried out with the desulfurization of alkylated dibenzothiophenes (Cx-DBTs) using the desulfurizing microorganism Mycobacterium sp. G3. In the water-phase reaction system, the desulfurization activities were constant with respect to species of Cx-DBTs as substrates. However, the desulfurization activities in the oil/water two-phase reaction system against DBT, 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT, and 4,6-dibutyl DBT were 49.0, 45.9, 11.5, 1.35, and 0.00 micromol g DCW(-1) h(-1), respectively. The kinetic parameters for the degradation of DBT, 4,6-dimethyl DBT, and 4,6-diethyl DBT were also obtained (V(max) values 90.0, 68.7, and 22.7 micromol g DCW(-1) h(-1) and K(m) values 0.21, 0.70, and 3.03 mM, respectively). The reason for the decrease in activity against Cx-DBTs of high molecular weight was a decrease in the V(max) value and an increase in the K(m) value, the latter being a particularly serious problem. Furthermore, the hydrophobicity of the substrate was evaluated as the capacity factor measured by high-performance liquid chromatography (HPLC). The correlation between substrate hydrophobicity and desulfurization activity indicated that the desulfurization reaction in the oil/water two-phase reaction system is greatly influenced by the hydrophobicity of the substrates. In addition, the influence of the solvent on desulfurization activity was examined, and it was found that not only the hydrophobicity of substrates, but also that of solvents, affected the desulfurization reaction.