One of the most dangerous endemic scorpions of Iran is Hemiscorpius lepturus that its venom is very toxic and known as responsible for most of scorpion sting dependent deaths in Iran. There are four Metalloproteinase inhibitors in the venom gland transcriptome of this scorpion (HLMetInhibit1-4). Metalloproteinase inhibitors are involved in some of the critical cells activity including the stability and surviving of ECM. This study aimed to investigate the structure and functions of a pair of these molecules and subsequently their cloning and expression for the first time.
Bioinformatics analysis and molecular docking on sequences of HLMetInhibit1 and HLMetInhibit2 performed (with the MG764541 and MG764542 NCBI accession numbers). The recombinant plasmids (pET-22b- HLMetInhibit 1,2) designed and synthesized, then transformed into the E. coli BL21 bacterial host. The colony-PCR and electrophoresis on one percent agarose gel performed and protein expression induced by different concentrations of IPTG at different times. Then SDS-PAGE and staining by coomassie brilliant blue and western blotting performed.
The highest binding affinity predicted for HLMetInhibit1 by molecular docking results. Gene cloning performed. Highest protein expression detected after 5 hours and with 1 mM concentration of IPTG. SDS-PAGE and western blotting confirmed the protein expression correctness for HLMetInhibit1 and HLMetInhibit2 with 26 and 17 KD of molecular weights.
Gene cloning and protein expression of Hemiscorpius lepturus metalloproteinase inhibitors 1 and 2 as the novel proteins that have not been studied so far, is approachable and it is hoped that after further in vitro and in vivo investigations, they will be the suitable candidates for research in the Various therapeutic fields.
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