Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition.
The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 mug/mL of expression culture were identified.
Blommel
Paul G
PG
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Becker
Katie J
KJ
Duvnjak
Petar
P
Fox
Brian G
BG
D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics*; D049453:Luciferases, Bacterial / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics
D003470:Culture Media; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D002244:Carbon; D049453:Luciferases, Bacterial; D010100:Oxygen