A comparison of inoculation methods to simplify recombinant protein expression screening in Escherichia coli.
In the past five years, Structural Genomics (SG) initiatives have established an automated pipeline for protein production in Escherichia coli to rapidly screen various conditions, resulting in soluble expression of recombinant proteins to aid in carrying out structural studies. However, some steps of the procedure are still extensive and require manual handling. Here, we present a comparative study of one step of the process, E. coli cultivation, using a set of 12 expression vectors encoding for fusion proteins of seven independent target proteins. First, we show that performing E. coli growth in auto-inducible medium (ZYM-5052) results in a comparable protein expression/solubility profile to that obtained when growing cells in classical Luria-Bertani (LB) medium. Second, we show that the transformation mix can be used directly to inoculate a culture, saving time and circumventing the error-prone step of colony picking, without impairing cell growth and the protein expression/solubility profile. Thus, we show that a basic, but nevertheless essential, step of a protein production pipeline, E. coli cultivation, can be simplified to a single event that is fully compatible with complete automation.
Busso
Didier
D
Institut de Génetique et de Biologie Moléculaire et Cellulaire/Centre National de Recherche Scientifique/Institut National de la Santé de la Recherche Médicale, Université Louis Pasteur, Illkirch, France. djbusso@igbmc.u-strasbg.fr
Stierlé
Matthieu
M
Thierry
Jean-Claude
JC
Moras
Dino
D
D000667:Ampicillin / Q000494:pharmacology; D002228:Carbenicillin / Q000494:pharmacology; D003470:Culture Media; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D013997:Time Factors; D014169:Transformation, Bacterial
D003470:Culture Media; D011994:Recombinant Proteins; D000667:Ampicillin; D002228:Carbenicillin