Site directed mutagenesis to produce mutant Iranian rice-NSLTP2 (Oriza sativa) protein that contains Y45Wmutation

NSLTPs are plant proteins that are subdivided into nsLTP1 (9kDa) and nsLTP2 (7kDa) according to the molecular weight. These highly stable proteins can protect drugs against oxidation or degradations. Rice NsLTP2 due to the inherent ability to cross biological membranes and bind to steroid compounds (including some pharmaceutical compounds) have potential application for use in drug delivery systems. After designing of mutagenic primers (contains a mutation of (Y45W)) the method of SOE-PCR (splicing by overlap extension-polymerase chain reaction) is used to site directed mutagenesis in the gene encoding the rice nsLTP2. Wild type nsLTP2 was cloned in to the PGEX6p2 vector. This wild type plasmid was used as a template for constructing the mutant fragment and designed mutant primers are used for amplification of mutant fragments. Mutant rice nsLTP2 are expressed in the pET32a vector in to the E. coli BL21 (DE3) strain. After the amplification of the mutant fragments and confirmation of mutations generated by sequencing، the mutant constructs cloned into the pET32a vector and the fusion mutant protein with a His-tag was purified by Ni-NTA Affinity chromatography column. The purpose of this study is Site directed mutagenesis to production the mutant Iranian rice-NSLTP2 (Oriza sativa) protein that contains Y45Wmutation. We hope that، in the future، these findings could contribute that this protein could be used to create biosensors or act as drug carriers.