Production and characterization of a recombinant single-chain antibody (scFv) for tracing the σ54 factor of Pseudomonas putida

The number of alternative sigma factor molecules per bacterial cell determines either stochasticity or evenness of transcription of cognate promoters. An approach for examining the abundance of sigmas in any sample of bacterial origin is explained here which relies on the production of a recombinant highly specific, high-affinity single-chain variable Fv domain (scFv) targeted towards unique protein sites of the factor. Purposely, a super-binder scFv recognizing a distinct epitope of the less abundant sigma σ54 of Pseudomonas putida (also known as σN) was obtained and its properties examined in detail. To this end, an scFv library was generated from mRNA extracted from lymphocytes of mice immunized with the purified σ54 protein of this bacterium. The library was displayed on a phage system and subjected to various rounds of panning with purified σ54 for capturing extreme binders. The resulting high-affinity anti-σ54 phage antibody (Phab) clone named C2 strongly attached a small region located between positions 172 and 183 of the primary amino acid sequence of σ54 that overlaps its core RNA polymerase-binding region. The purified scFv-C2 detected minute amounts of σ54 in whole cell protein extracts not only of P. putida but also Escherichia coli cells and putatively in other bacteria as well. The affinity constant of the purified antibody was measured by surface plasmon resonance (SPR) and found to have a KD (koff/kon) in the range of 2 × 10−9 M. The considerable affinity and specificity of this recombinant antibody makes it a tool of choice for quantitative studies on gene expression of σ54-dependent promoters in P. putida and other Gram-negative bacteria.

► Recombinant single-chain antibody technology has proven instrumental for isolating a highly specific, high affinity scFv that binds distinctively the sigma 54 factor of Pseudomonas putida.
► The site of the sigma factor recognized by the antibody encompasses a minimal sequence of 10–12 amino acid residues which bind scFv C2 both as an isolated linear epitope and in the context of the full-size protein.
► scFv C2 interacts with the part of sigma 54 believed to have direct interaction with the RNA polymerase.
► The antibody reported in this work is an invaluable tool for quantifying the number of sigma molecules per cell of P. putida growing under various conditions.