PROTEIN EXPRESSION AND PURIFICATION 8, 17–22 (1996) ARTICLE NO. 0069 Overproduction and Purification of s S , the Escherichia coli Stationary Phase Specific Sigma Transcription Factor Lam H. Nguyen and Richard R. Burgess 1 McArdle Laboratory for Cancer Research, University of Wisconsin – Madison, Madison, Wisconsin 53706 Received January 8, 1996, and in revised form March 1, 1996 shown directly to be a s factor based on biochemical This paper reports the overproduction and the de- studies. It binds to and confers promoter specificity on tails of a rapid method to purify active s S monomers core RNA polymerase (4,9). from a T7 RNA polymerase-based protein expression Any detailed studies of the structure and functions system. This 2-day procedure involves solubilizing in- of a particular protein require that the protein be avail- clusion bodies in sarkosyl detergent, removal of sarko- able in reasonable amounts. We present in this report syl by dialysis, and a single gel filtration column chro- the details of a rapid procedure for obtaining pure and matography step. The final yield of s S is about 9 mg of active monomeric s S in large quantities for physical approximately 92% purity from 0.5 g of wet weight and biochemical characterizations. cells. Overproduced s S binds to core RNA polymerase and supports transcription from the bolAp1 promoter, MATERIAL AND METHODS a s S -dependent promoter.  1996 Academic Press, Inc. Plasmid constructions and cell strains. Various plasmid constructs are summarized in Table 1. A DNA fragment that contains the amino-terminal part of the Escherichia coli DNA-dependent RNA polymerase s S protein coding region was made by polymerase chain holoenzyme consists of a core polymerase (a 2 bbv) and reaction (PCR) with conditions as described (10) using a sigma (s) subunit (1). The presence of a s factor pMMKATF2 (11) as the template and the follow- allows RNA polymerase to initiate transcription at spe- ing primers: the forward primer is 5-CGGGAT- cific DNA sequences called promoters. There are sev- CCATATGAGTCAGAATAC-3 (with the first codon eral s factors in E. coli, each of which directs transcrip- underlined) hybridizing to base 1 to 14 and the reverse tion of distinct promoters when bound to core RNA primer is 5-ATATCGTCATCTTGCGTGG-3 hybridiz- polymerase (2). ing to base 746 to 764 of the s S DNA coding sequence. When E. coli cells enter the stationary phase of The s S coding sequence is 1053 base pairs long (11) growth, they undergo complex physiological and mor- and codes for a 40,038-dalton protein. This PCR DNA phological changes. Accordingly, there are transient fragment was digested with AccI and BamHI and changes in the rate of expression of a large number of cloned into the same sites in pSP72 (Promega). This stationary-phase/starvation genes. Most of these sta- construct, pLHN26, was sequenced from the BamHI to tionary-phase/starvation genes are dependent on the an EagI site within the s S gene in order to check for s S gene rpoS/katF (3). The katF/rpoS gene product s S possible PCR errors due to Taq DNA polymerase misin- increases by at least an order of magnitude during corporation. None was found. An EagI– HincII frag- transition from exponential to stationary phase (4). s S ment of pMMKATF2 (11) that contains the rest of the synthesis has also been shown to be positively regu- s S gene was then cloned into pLHN26 at the same lated by the level of ppGpp (5) and homoserine lactone restriction sites in order to replace the rest of the PCR (6). Also, s S synthesis is negatively regulated by UDP – s S DNA sequence. This construct is called pLHN29. glucose (7) and cAMP (8). The cellular concentration A NdeI– XhoI (filled-in with Klenow DNA polymerase) of s S is controlled at the levels of transcription, transla- fragment from pLHN29 that has the s S gene was tion, and protein stability (8). Recently, s S has been cloned into pET11T overexpression vector (12) at the NdeI and blunted BamHI sites. This final plasmid con- struct is called pLHN30. All plasmid constructs were 1 To whom correspondence should be addressed. 17 1046-5928/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.