Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Fine bubble mixing (FBM) culture of E. coli: a highly cost-effective middle scale-size culture system.

A highly cost-effective and easy-to-assemble cultivation system suitable for middle scale-size culturing of bacterial cells is described. In the culture, from a flat-shaped air-stone with large surface area, fine bubbles are generated with a low-cost air pump available in an aquarium fish shop, and cell-agitation and oxygen supply are efficiently conducted by fine bubbles simultaneously. Growth properties of the cells and their saturation density are comparable to those in a conventional culture system. The expression of recombinant protein was revealed to be similar to conventional methods. The system does not require any expensive machines or equipments. In addition, all equipments except plastic flat-shaped airstone are reusable after sterilization. Due to the low cost, the ease to use and multiple cultivations at once, our system may enable to find better culture conditions, to scale-up with ease and to perform timesaving efficient protein production.