Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification.
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 β-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%.
Department of Biology Engineering, Beijing Polytechnics, Beijing 100029, P.R. China.
Hu-Cheng
HC
Zhang
Department of Biology Engineering, Beijing Polytechnics, Beijing 100029, P.R. China.
Jun
J
Yang
Department of Biology Engineering, Beijing Polytechnics, Beijing 100029, P.R. China.
Guo-Wei
GW
Yang
Department of Biology Engineering, Beijing Polytechnics, Beijing 100029, P.R. China.
Xiao-Jie
XJ
Wang
Department of Biology Engineering, Beijing Polytechnics, Beijing 100029, P.R. China.
Hai-Tao
HT
Fan
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013010:Sonication; D013862:Thiogalactosides / Q000494:pharmacology
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011994:Recombinant Proteins; D013862:Thiogalactosides