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 β‐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.

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Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to s 1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors. ß

not available DS (Child) H J 2021; 37(2): 87-92

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