Introduction

The tobacco etch virus (TEV) protease provides an in vivo tool to study protein topology, by identifying exposed versus buried residues or intracellular versus extracellular domains of membrane proteins. The TEV protease recognizes a rare seven amino acid sequence glu-asp-leutyr-phe-gln-ser and cleaves between the gln-ser residues (1). Previous research suggested that insertion of the recognition site does not interfere with protein function due to its small size (2). This led to the exploitation of TEV protease as a tool for dissecting functional domains of proteins.

Ehrmann et al. (2) designed an elegant transposon-based approach for insertion of TEV cleavage sites (TEVcs) into multiple sites in a gene. With a large number of insertions, this approach can provide useful information on the topology of certain proteins in vivo. However, it does not allow directed insertions of the TEVcs at defined sites to test specific predictions about protein topology. We developed an alternative method for rapidly constructing insertions at defined sites in an open reading frame (ORF). This approach is based upon three genetic tricks: (i) use of the λ Red recombination system to construct directed insertions of a selectable marker; (ii) use of the λ Red recombination system to replace the selectable marker with a short, nonselectable sequence; and (iii) use of an in-frame, downstream reporter to enrich for replacements of the desired type.

The λ Red recombination system provides an efficient genetic tool for introducing TEVcs insertions at specific sites in a gene. Although Red-mediated genetic exchange is specific and efficient, strong counterselections for insertion of seamless mutations are limited. When used for such singlecopy substitutions, commonly used counterselectable markers yield false positive results so frequently that isolation of desired mutants demands labor-intensive secondary screening. In contrast, placement of an in-frame copy of the lac operon under transcriptional control of the target gene provides a clean and robust counterselection (Figure 1). The lac insertion also provides a tool for monitoring the effect of the insertions on transcription. Coupling these two genetic tricks provides an efficient approach to introduce seamless, in-frame insertions of the TEVcs into a specific location in the target gene. Once TEVcs insertions are confirmed, TEV protease cleavage assays can be done in vivo or with purified protein in vitro to determine the accessibility of TEVcs in the protein.

Figure 1. The λ Red system was used to insert the tobacco etch virus cleavage site (TEVcs) into putA through the use of a two-swap method. (Click to enlarge)

We tested this method on the PutA protein from Salmonella enterica sv. Typhimurium. PutA is a multifunctional protein involved in both proline degradation and autogenous regulation of the put operon. In the absence of proline, PutA dimerizes and binds DNA to repress transcription of the put operon. In the presence of proline, PutA associates with the membrane and catalyzes the oxidation of proline to glutamate. While a great deal is known about the enzymatic activities of PutA, little is known of PutA topology, and few distinct domains have yet been assigned to these activities. Recently, the crystal structure of a truncated form of the Escherichia coli PutA (PutA₆₆₉) protein has been solved, including the 669 amino acids of the N terminus (3). The crystal structure of PutA₆₆₉ led to predictions of domain organization. Insertion of TEVcs into PutA allowed tests of some of these predictions in vivo.

Materials and Methods

Strains and Media

When required, antibiotic concentrations in rich media were as follows: 90 µg/mL ampicillin, 20 µg/mL chloramphenicol, and 50 µg/mL kanamycin (all from Sigma, St. Louis, MO, USA). In minimal media, a lower concentration of ampicillin (15 µg/mL) was used. Minimal media were prepared with sodium succinate as a carbon source supplemented with or without 0.2% proline. Lactose plates were prepared as no citrate E (NCE) media plates supplemented with 0.4% lactose (4).

Construction of a π-Dependent Vector for Use as a PCR Template

In order to produce a template for PCR amplification, the TEVcs was cloned into π-dependent plasmid pGP704 (5). The TEVcs was created by annealing oligonucleotides of the corresponding amino acid sequence of a wild-type cleavage site. Complementary oligonucleotides were designed with flanking EcoRI and XbaI sticky ends as follows: 5′-CTAGAGAAAACCTGTATTTTCAGAGCG-3′ and 5′-AATTCGCTCTGAAAATACAGGTTTTCT-3′. pGP704 was digested with XbaI and EcoRI (Fermentas, Hanover, MD, USA) according to manufacturer's recommendations. Ligation of pGP704 and the TEVcs yielded plasmid pPC265.