In vitro expression of the recombinant TLP protein from Pinus sylvestris and study of its antimicrobial activity

One of the most important inducible defense mechanisms is the biosynthesis of pathogenesis – related (PR) proteins. Members of the PR – 5 groups are called thaumatin – like proteins (TLPs) because their amino acid sequences are homologous to thaumatin, a sweet – tasting protein from the West African shrub Taumatococcus danielli. Many PR – 5 proteins are induced in plants in response to infection by pathogens, osmotic stress, treatment with abscisic acid, ethylene, salicylic acid and wounding. Members of this group have been shown to have antifungal activity against a broad spectrum of fungal pathogens. Previous results from our laboratory have shown that TLP gene expression increases in P. sylvestris after inoculation with Heterobasidion annosum; therefore the effect of TLP protein on growth of H. annosum was studied. The Pinus taeda TLP gene sequence was used to design primers for amplification of the full-length TLP gene from P. sylvestris (PsTLP). The full-length PsTLP has an open reading frame of 606 bp, encoding a protein of 202 amino acids, molecular weight approximately 22.5 kDa, including 16 cysteine residues which are characteristic of L-type TLPs. The disulfide bridges formed by these cysteine residues have important role in maintaining the protein stability and correct folding and preserving high activity under extreme temperatures and pH conditions. In addition, the deduced protein has a thaumatin family signature G-x-[GF]-x-C-x-T-[GA]-D-C-x(1,2)-[GQ]-x(2,3)C. Also sequence has 5 conserved amino acids residues responsible for the acidic cleft with known antifungal activity. This conserved acidic cleft is comprised of five amino acids (R, E, D, D, D) and is believed to be involved in binding to β-1,3-glucan on the fungal cell membrane, resulting in a targeted interaction between host TLP and the fungal cell, which leads to permeabilization of the fungal cell membrane and disruption of the osmotic balance inside hyphal cells, resulting in cell rupture. To study the effect of Pinus sylvestris TLP protein on fungal growth, the coding sequence was expressed in vitro in a cell – free expression system. The advantages of in vitro translation system include time saving, the possibility to produce proteins that are toxic and the possibility of using PCR products as templates for protein synthesis. This greatly accelerates the protein production process, because no cloning steps are required. In order to examine the effect of the in vitro expressed TLP, an agar plug containing mycelia of H. annosum was placed in the center of agar plates and incubated for 1 week. Th e PsTLP at various concentrations were applied to sterile fi lter paper discs surrounding the plugs 1 cm away from the rim of the mycelia colony. Aft er 2 weeks incubation at 25oC, no clear inhibition zones were seen but in the plate with 100 μl protein, H. annosum mycelia were lighter and thinner than in the plate with 15 μl of protein. Work is continuing to obtain suffi cient amounts of protein for more eff ective antifungal activity assay and to determine if the expressed His tag may have a negative eff ect on protein activity. Also, the protein may have incorrect folding or be without necessary posttranslational modifi cations which the in vitro expression system cannot introduce.