Expression, Purification And Preliminary Antibody Preparation Of Sls1p Of Saccharomyces Cerevisiae

Mitochondria are described as "cellular power plants". The instability of mitochondrial genome would lead to the deficiencies of oxidative phosphorylation and ATP production, further affect the metabolism, free radicals and apoptosis of the cell. Systematic Name of SLS1(Sigma Like Sequence) is YLR139C, which locates from421543to423474of chromosome Ⅻ of Saccharomyces cerevisiae. SLS1contains an open reading frame (ORF) of1929bp, and encodes a mitochondrial protein of643amino acids with a calculated molecular mass of73.2kDa and an isoelectric point of10.02. It has been reported that Slslp could coordinate transcription and translation through Nam1p.We found that the slsl null mutant strain was unable to grow in glycerol medium and showed obviously decrease in total ATP level. Mitochondrial genome’s stability decreased in slsl null mutant strain. The content of eight mtDNA genes including COX1, COX2, COX3, ATP6, ATP9, CYTB,15S rRNA and21S rRNA decreased significantly and differently. However, the content of eight mtDNA genes increased significantly and differently in SLS1restoring expression and over-expression strains. These results showed that SLS1was related to the stability of mtDNA. The mechanism of SLS1influencing the stability of mtDNA is still not clear, and the interactions of Slslp with other mitochondrial nucleoid-associated proteins have not been reported. There is no antibody of Slslp for sale, so we will construct fusion expression vector pET2S-SLS1, express, purify protein and prepare monoclonal antibody for researching the interaction of Slslp and other mitochondrial nucleoid-associated proteins. In order to obtain over-expressed Slslp, PCR was used for amplifying the gene SLS1from the chromosome of Saccharomyces cerevisiae. Then the PCR fragment was cloned into the expression vector pET28a to get recombinant plasmid (pET28a-SLS1). The positive recombinant, verified by restriction endonuclease digestion and sequence analysis, was transformed into E. coli BL21(DE3). The engineering strain expressing proteins was over-expressed so as to find the optimized induced condition:induced4h at37℃with0.4mmol/L IPTG. The relative amount of product was54%, mainly in the form of inclusion body. After inclusion body was dissolved via8mmol/L urea, the protein was purified by the Ni+affinity chromatography. The concentration of purified protein was2.5mg/mL and the purity was88.3%. The monoclonal antibody was preliminarily prepared by immunizing Slslp by Western Blot. It will not only set foundation for researching the interactions between Slslp and other mitochondrial nucleoid-associated proteins, but also its function in the stability of mitochondrial genome.