Preparation of uniformly labeled NMR samples in Escherichia coli under the tight control of the araBAD promoter: expression of an archaeal homolog of the RNase P Rpp29 protein.
We report the first use of the tightly regulated araBAD promoter for generating uniformly labeled samples for NMR. The araBAD promoter provides a distinct advantage over that of the most commonly used protein overexpression systems in bacteria (e.g., in pET vectors: T7lac), in that it provides much tighter control over basal expression. However, use of araBAD-regulated expression for preparation of uniformly labeled protein samples for NMR is complicated by the fact that glucose (the most commonly used carbon source in defined minimal media) indirectly represses transcription, and thus, cannot be used. After experimenting with alternative media, we found that uniformly labeled NMR samples can be prepared using the highly regulated arabinose-inducible promoter and that suitable yields can be obtained in defined minimal media containing glycerol, not glucose, as the carbon source.
Boomershine
William P
WP
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Raj
M L Stephen
ML
Gopalan
Venkat
V
Foster
Mark P
MP
D019747:Aldose-Ketose Isomerases / Q000235:genetics; D001089:Arabinose / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D001089:Arabinose; D017853:Phosphotransferases (Alcohol Group Acceptor); C022550:ribulokinase; D043262:Ribonuclease P; D002238:Carbohydrate Epimerases; C022554:L-ribulosephosphate 4-epimerase; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase