Production and purification of recombinant SMN1 protein for using in the treatment of SMA disease

Spinal Muscular Atrophy is a disease characterized by muscle weakness and atrophy caused by permanent deterioration of the anterior horn cells in the spinal cord. Affecting one child in 10000 births, autosomal recessive SMA is the most common genetic cause of infant mortality. SMA is caused by defects in the SMN1 gene. There are 4 types of SMA. Treatment strategies are very limited and very expensive in the world. In this thesis study, recombinant SMN1 protein will be created together with TAT peptide and its drug-forming potential will be evaluated in vitro. The studies consist of 3 work packages. Firstly, SMN1 gene was cloned from pEGFP/N2 vector by restriction enzymes and cloned into pET-21a vector. This clone was named SMN1-pET-21a. In the second stage, cell transit protein sequences (TAT sequence) were cloned next to this clone to enable the SMN1 protein to pass through the cell membrane. This clone was named as TAT-SMN1-pET21-a. The empty pET-21a plasmid was used as a control. All plasmids were transformed into E.coli BL21(DE)3 bacterial strains. In the second work package, E.coli BL21(DE)3 bacteria were induced with IPTG. TAT-SMN protein with histidine tail was then purified from the protein lysate produced by Ni-Nta method. Purification was confirmed by Western blot and SDS-Page. In the third work package, SMN1 gene expression in SH-SY5Y cells was silenced by lentiviral pLKO1 plasmid and shRNA system to examine the effect of recombinant protein activity under in vitro conditions. After single colony selection, silencing was confirmed at the mRNA and protein level. Then, the purified recombinant protein was applied to the cells at different concentrations and times. The effect of the recombinant protein on cell viability was determined by MTT assay. Whether it enters into the cell was determined using immunofluorescence technique. Then, SH-SY5Y and SMN1 expression silenced cells were treated with recombinant TAT-SMN protein at different concentrations and SMN protein levels in the cells were determined by immunofluorescence technique. The produced drug candidate was found to be successful in in vitro activity assays.