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 William P. Boomershine, a M.L. Stephen Raj, b Venkat Gopalan, a,b,c and Mark P. Foster a,b,c, * a Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA b Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA c Protein Research Group, The Ohio State University, Columbus, OH 43210, USA Received 27 August 2002, and in revised form 19 November 2002 Abstract 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 ex- perimenting 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: argU; E. coli; Glycerol minimal medium; Isotope labeling; NMR; Overexpression; pBAD; pET; RNase P; T7 RNA polymerase. Ongoing structural genomics efforts rely heavily on heterologous overexpression of all genome encoded proteinsinbacterialhosts[1].Currently,themostwidely used approach involves the cloning of a target gene under the control of bacteriophage T7 transcription and translation signals, and subsequent expression of the protein in a host Escherichia coli strain that can supply T7 RNA polymerase [2,3]. Inducible expression is achieved by using an IPTG-inducible lacUV5 promoter toregulateexpressionofthechromosomallyencodedT7 RNA polymerase. Basal (leaky) transcription of the lacUV5-regulated T7 RNA polymerase, and consequently, the gene of interest, is deleterious to efficient overexpression of proteins; leaky expression is particularly problematic when the desired proteins are toxic to the host. In many of the commercially available vectors (e.g., pET-15b, Novagen), the target gene is cloned under the control of a T7lac promoter, which contains a 25-bp lac operator immediately downstream from the 17-bp T7 promoter [2,3]. This provides an additional level of control over basal expression, since the lac repressor binds to the T7lac promoter on the plasmid and inhibits transcrip- tion by T7 RNA polymerase. Repression could also be achieved by co-expressing T7 lysozyme, an inhibitor of T7 RNA polymerase, from a plasmid (pLysS/pLysE) [3,4], requiring the use of a second antibiotic to avoid loss of this plasmid. An alternative approach to prevent undesirable basal expression exploits the tight regulation offered by the araBAD promoter of the arabinose operon [5]. Tran- scriptional regulation is accomplished by AraC, a pro- tein that exerts negative and positive control in the absence and presence of L -arabinose, respectively [6,7]. Target genes cloned under the control of araBAD Protein Expression and Purification 28 (2003) 246–251 www.elsevier.com/locate/yprep * Corresponding author. Fax: +614-292-6773. E-mail address: Foster.281@osu.edu (M.P. Foster). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-5928(02)00707-6