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

Abstract

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 σ(54) of Pseudomonas putida (also known as σ(N)) 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 σ(54) protein of this bacterium. The library was displayed on a phage system and subjected to various rounds of panning with purified σ(54) for capturing extreme binders. The resulting high-affinity anti-σ(54) phage antibody (Phab) clone named C2 strongly attached a small region located between positions 172 and 183 of the primary amino acid sequence of σ(54) that overlaps its core RNA polymerase-binding region. The purified scFv-C2 detected minute amounts of σ(54) 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 K(D) (k(off)/k(on)) 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 σ(54)-dependent promoters in P. putida and other Gram-negative bacteria.