Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags.

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.

Functional domains of the TOL plasmid transcription factor XylS.

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence.

XylS, an AraC family transcription factor, positively regulates transcription of Pseudomonas putida TOL plasmid meta operon from the Pm promoter. A tandem of 15 bp homologous direct repeats, separated by 6 bp and overlapping with the -35 hexamer of the promoter, is required for the activation of Pm by XylS in vivo. In this study we have characterized specific binding of XylS to the Pm operator Om. XylS was overexpressed with an epitope tag in its N-terminus. Tagged XylS (N-XylS) was immunopurified and was shown to specifically bind to Om. We have used matrix-bound N-XylS in DNA footprinting and methylation interference experiments. Binding of N-XylS protects 44 bp in the Om region on both strands from DNase I digestion and generates hypersensitive sites (within the protected area) which lie on the same face of the DNA helix. Results of hydroxyl radical footprinting and methylation interference assays indicate that XylS binds along one side of the DNA and covers four helical turns. The protein has base-specific contacts in four adjacent major groove regions on the same helical face. Our data are in accord with the prediction of the presence of two separate DNA-binding units in an XylS molecule which are involved in base-specific contacts in two adjacent major-groove regions of a half-site. The direct repeat arrangement of the binding site and the mode of DNA binding of XylS are similar to the arrangement of recognition sites and the DNA contact pattern of AraC protein from Escherichia coli.