Cloning,Expression And Purification Of MIS Complex Proteins

In most eukaryotes,N6-methyladenosine(m6A)is the most prevalent internal modification which occurs in messenger RNA(mRNA)and long noncoding RNA(lncRNA).It has functions in lots of RNA metabolism pathways(mRNA splicing,translation and degradation).m6A modification plays a vital role in gene expression regulation.It is involed in different physiological process(eg.spermatogenesis,embryo development,and aopotosis).The m6A modification,which is reversible and dynamic,is co-regulated by methytransferases and demethylases.In Saccharomyces cerevisiae,the methyltransferase complex consists of MUM2,IME4 and SLZ1(MIS complex).IME4 has methyltransferase activity and MUM2 may activate the MIS complex by activating the catalytic activity of IME4 or by targeting IME4 to mRNA;Slzl may have regulatory functions.In order to study the molecular mechanism of m6A,research on MIS complex is necessary.PCR and sequencing results confirmed the successful cloning of the target gene into the vector.The expression of protein was induced by IPTG and high concentration of the recombinant protein was obtained via the purification process by affinity-chromatography,gel filtration chromatography or ion exchange chromatography.Therefore,we produced MIS complex proteins from E.coli for preparation of large quantity for structure function studiesConstruction of the recombinant plasmid:The S.cerevisiae genes encoding different fragments of MUM2,IME4 and SLZ1 proteins were amplified by polymerase chain reaction(PCR)from Mammalian Gene Collection(MGC),and sub-cloned in prokaryotic expression vector pET28a-MHL.PCR and sequencing results confirmed the 15 successful recombinant plasmids.Expression of recombinant MIS complex proteins:The positive recombinant plasmids were transformed into the host,E.coli BL21(DE3)and E.coli Rosetta(DE3).The addition of IPTG induced the overexpression of recombinant proteins.Our data showed:soluble form expression(8 recombinant proteins),inclusion body form expression(4 recombinant proteins).Optimization of expression conditions:Expression conditions(induction time,induction temperature and IPTG concentration)were optimized by using single factor experiment.Different proteins have different optimal expression conditions.Purification of recombinant MIS complex proteins:Expressed proteins(IME41-600,IME41-110,MUM21-366,MUM2180-366,SLZ12-298,SLZ12-129)were purified by affinity chromatography or gel filtration chromatography.Among them,various buffers were used to solubilize IME41-600 from the inclusion body.Target proteins with high purity and high stability had been obtained.