Shinichi Fukuzono

Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring

Abstract Fed-batch cultures of recombinant Escherichia coli HB101 were investigated to obtain high cell density and large amounts of β-galactosidase (β-gal). E. coli HB1010 was transformed with a hybrid plasmid pTREZ1, which contained a β-gal gene controlled by the trp promoter. In fed-batch cultures of recombinant E. coli , when the cell concentration reached around 13 g / l , the cell growth stopped and large amounts of inhibitory substances have accumulated in the broth. These inhibitory substances were isolated and identified. Acetate produced by the cells was evidently the main inhibitor of cell growth and β-gal production. Since the cells proved to assimilate acetate, the feed rate was controlled with acetate concentration monitoring in the fed-batch culture. As a result, the acetate concentration was maintained at a low level and cells grew smoothly without acetate-induced inhibition. Cell concentration and β-gal quantity reached high values of 28 g / l and 64 U/ml, respectively.

Cultivation of Escherichia coli Harbouring Hybrid Plasmids

Abstract Cultivation of Escherichia coli transformed with a hybrid plasmid pTREZ1, containing a β-galactosidase (β-gal) gene under the control of the trp promoter, was investigated to develop a control method for effective production of heterologous proteins in bacteria. When glucose in the culture broth was consumed, dissolved oxygen (DO) concentration and respiratory quotient (RQ) rapidly increased. Then the inducer, 3-β-indolylacrylic acid, and casamino acids were added to induce the trp operon. β-Gal production per cell mass reached about 5 U/mg. In addition, RQ rapidly increased when β-gel production ceased, and proved to be a better indicator of E. coli cultivation than DO concentration.

Expression of the histamine receptor in Xenopus oocyte and its application to the histamine sensor

Abstract We developed a receptor-mediated biological sensor for real time detection of histamine derived from granules of mast cell and basophils. A human histamine receptor gene was cloned by polymerase chain reaction and its mRNA was injected into Xenopus oocyte. After incubation for 2–3 days, the histamine receptor was expressed in the cell membrane of the oocyte. When the oocyte was activated by histamine, a calcium-activated chloride channel was opened, and the chloride ions in the oocyte flowed to the outside. The chloride ion current intensity was correlated with the histamine concentration. The histamine sensitivity of the oocyte was sufficient to allow comparison with other methods histamine analyses, such as enzyme immunoassay or HPLC analysis. The oocyte sensor system described here is a new chemical sensor system using receptor-mediated biological reactions.