An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remai...An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.展开更多
基金This work was supported by the National Science Foundation CBET Division CAREER Award(grant number:1254148)the Utah NASA Space Grant Consortiumand by the Simmons Center for Cancer Research at Brigham Young University.
文摘An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.
