P-50: Expression of nsLTP Drug Protein in E.coli (Pages: 62-63)

Karimian M *, Miroliaie M , Zahraie Z ,


Objective: Non specific lipid transfer proteins (nsLTPs) are a family of group proteins with low average molecular weight in plants that have potential applications especially in drug delivery systems.Based on their molecular weight LTPs could be categorized into two classes: LTP1 and LTP2. Many active agents used in pharmaceutics and cosmetics need a protective shielding from sensitive environment. This can be achieved by using carrier systems. nsLTPs have the high resistance to denaturants, heat and proteases therefore can bind a broad range of lipid molecules are potential as drug carriers. The aim of this research was cloning and expression of nsltp2 in E.coli. Materials and Methods: Cloning and construction of nsltp2 Rice genome was extracted from fresh leave by CTAB method. Rice nsltp2 has no intron therefore to design specific primers, the entire genomic sequences of rice nsltp2 was deduced from NCBI and analyzed with oligo6 software. Specific primers pair were designed introducing BamH I and XhoI sites at 5´ end of forward and reverse primers respectively. Both of fragment containing amplified nsltp2 gene and vector were treated with BamHI and XhoI to produce the sticky ends for ligation. Finally the BamHI- XhoI fragment containing the entire coding sequence of nsltp2 inserted in to the same place in pGEX-6p-2 and preformed ligation. Propagation of recombinant plasmid was performed by transforming into one shot TOP10 E.coli cells. At the next day grown colonies were checked by colony PCR using the vector specific primers. Further recombinant plasmid was subsequently were send to sequence. SDS-PAGE analysis of bacterial lysate cells Transformed E.coli cells were cultured in 50 ml of LB media. Then protein expression was induced by adding IPTG. Sonication method was implemented to yield cell ruptures as described elsewhere. At the next step 1/100 volume of lysate cells were subjected to SDS-PAGE and CBB staining Results Amplification of nsltp2 At the end of PCR was produced 300bp DNA fragment that was the same with nsltp2 gene length. Sequencing of nsltp2 Data from sequencing analysis confirmed nsltp2 sequence. Analysis of nsLTP expression with SDS-PAGE Data from SDS-PAGE analysis revealed that GST-nsLTP2 express in E.coli. Conclusion: Chao-Sheng et al. in 2004, evaluated applications of nsLTP in drug delivery system. Construction of PGEX-6p-2/nsltp2 expression vector Provides the field for subsequent research as to create site directed mutagenesis in nsltp, and whereby changes in the structure of nsLTP to promote applications in drug delivery systems