ABSTRACT
Recombinant Streptococcus Protein G (PG) is a cell wall protein, which, when combined with mammal immunoglobulin, is used in separating antibody technology. High-density fermentation technologies using an engineered recombinant PG-producing bacteria as well as PG separation and purification technologies have a direct impact on the availability and application of PG. Through primary and secondary seed cultivation, a recombinant E. coli strain was subjected to high-density fermentation with controlled feed supplement concentration under stimulation with isopropyl ß-D-1-thiogalactopyranoside. The present study investigated the effect of factors including inoculum size, oxygen levels, pH and the cultivating method on the fermentation process, as well as the effect of the separation and purification technologies, including ultrasonication, nickel column affinity chromatography, Sephadex G-25 gel filtration chromatography and diethylaminoethanol-sepharose fast flow ion exchange chromatography on the yield and purity of PG. The efficiency of extraction was detected using SDS-PAGE. High-density fermentation yielded 80-150 g/l of bacteria and 1 g PG was obtained from one liter broth. The present study delivered a highly efficient novel method via which PG can be obtained at a high concentration and a purity >95%.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Escherichia coli/metabolism , Fermentation , Gene Expression , Genetic Engineering , Hydrogen-Ion Concentration , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sonication , Thiogalactosides/pharmacologyABSTRACT
In this paper, microbial transformation of norkurarinone (1) by Cunninghamella blakesleana AS 3.970 was investigated and seven transformed products were isolated and characterized as kurarinone (2), 4â³,5â³-dihydroxykurarinone (3), 6â³-hydroxyl-2'-methoxyl-norkurarinone 7-O-ß-d-glucoside (4), 6â³-hydroxyl-norkurarinone 4'-O-ß-d-glucoside (5), 4â³,5â³-dihydroxynorkurarinone (6), 7-methoxyl-norkurarinone (7), and 7-methoxyl-4â³,5â³-dihydroxynorkurarinone (8), respectively. Among them, 3-5 are new compounds, and the glycosylation reaction in microbial transformation process was reported rarely. In addition, the cytotoxicities of transformed products (1-8) were also investigated.
