反应详情

The secretory expression system of the protransglutaminase using C. glutamicum has already been reported (WO01/23591). Then, by using the plasmid vector pPKSPTG1 for secretory expression of protransglutaminase described in WO01/23591, each of the YDK010 strain and the YDK010ΔPBP1a strain was transformed. Each of the obtained transformants was cultured in the MM liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogenphosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, and 50 g of calcium carbonate in a volume of 1 L with water, adjusted to pH 7.0) containing 25 mg/l of kanamycin at 30° C. for 72 hours. After completion of the culture, culture supernatant obtained by centrifuging each culture broth was subjected to reduced SDS-PAGE, and then stained with CBB R250 (produced by Bio-Rad). Secretion amounts of the protransglutaminase were determined according to the previous report (Protein Expr. Purif., 26:329-335), and the amounts were compared. As a result, the secretion amount of the protransglutaminase was significantly improved in the YDK010ΔPBP1a strain compared with that observed for the parent strain YDK010 (FIG. 8).

1. customAfter completion of the culture, culture supernatant obtained