Cloning and expression of the recombinant NP24I protein from tomato fruit and study of its antimicrobial activity
Thaumatin-like proteins (TLPs) constitutes a homogeneous family, members of which are produced by plants in response to different kinds of stress. NP24 protein is one of such salt-induced protein from tomato (Solanum lycopersicum) and it belongs to TLPs family. NP24 is a 24 kDa (207 amino acid) antimicrobial TLP found in tomato fruits. One isoform (NP24I) of NP24 was discovered in the outer pericarp of tomato fruit and is reported to play a possible role in ripening of the fruit in addition to its antimicrobial activity. In this study, the total RNA was isolated successfully from the outer pericarp of ripe (red) tomato fruit. cDNA was prepared and the gene coding for NP24I protein was amplified using conventional polymerase chain reaction (PCR). The gene was then cloned into Mach1™- T1® Escherichia coli cells, then subcloned into the over-expression vector pET-28a (+) using BL21 expression bacteria. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the gene was over-expressed in E. coli as inclusion bodies. Optimization of the recombinant NP24I protein solubility was achieved by cold induction through decreasing both expression temperature and Isopropyl-beta-thio galactopyranoside (IPTG) concentration. The recombinant NP24I protein was purified using Ni-NTA resin, and then the antimicrobial activity of the purified recombinant NP24I protein was tested. The aims of this work were to study the cloning and expression of NP24 protein from local tomato cultivar in a prokaryotic system and to test the activity of the recombinant NP24, as well as to prove the activity of native protein on the bacterial as well as fungal growth.
Key words: Pathogenesis-related proteins (PR), low molecular weight thaumatin-like protein (TLPs), cloning, Escherichia coli, cold induction, antimicrobial activity, tomato (Solanum lycopersicum).