Contruction Of Protein Sumoylation System In Prokaryotic Cell

Sumo is Small Ubiquitin-Related Modifier, named because of similar space structureand mode of action to ubiquitin. Sumo exits in eucaryon commonly and it has regulatingeffect to gene expression, damage repair and recombination of chromosome, celluarlocalization. The process of protein Sumo modification is reversible. Under the condition thatATP provides the energe, Sumo is actived after cleaving by protease and catalyzed byE1（Sae1-Sae2heterodimer）,E2（Ubc9）,E3（not needed in vitro experiment）,it makes proteinmodificated by the covalent bond connected to the substrate.The experiment makes Escherichia coli as the reaction container. Sumo modificationsystem is constructed by transfecting expression vectors containing E1,E2and sumo intoE.coli. Oct4was transfected into the system to detect the function of the system, then, theSumoylated Oct4was got.It is convenient to obtain Sumoylated protein by this system, theSUMOylation modification system constructed by us based on E.coli,laied a foundation for researching thesumoylation of proteins.（1）Construction of pGEX-Oct4、 pGEX-Oct4^k118R、 pCDF-Sae2-Sae1、pACYC-Ubc9-Sumo1expression vectors:The specific primers were designed with Sae1、Sae2、Ubc9、Sumo1and Oct4sequences provide byNCBI;Total RNA was extract from the teratoma cell F9using method of Trizol and wasreverse transcripted to cDNA,it was got the object fragments to make the cDNA as templateby PCR amplification. The expression vectors pCDF-Sae2-Sae1and pACYC-Ubc9-Sumo1were constructed. The substrate we selected of Sumo is Oct4which mutation is Oct4^k118Rasthe control of Oct4.The vectors of pGEX-Oct4and pGEX-Oct4^k118Rwhich have GST tagwere constructed.By sequencing,the vectors were correctly constructed.（2）The expression of proteins:The correct recombinate plasmids were transfected intocompetent cell to get expression bacterium.The expression bacterium were induced by1mmol/L IPTG at28℃, then they were collected and detected by SDS-PAGE orWesternBlot.We have detected the Sae1,Ubc9and Sumo1proteins by SDS-PAGE and Sae2protein by WesternBlot.The protein of Oct4and Oct4^k118Rwere also collectly expressed.（3）Construction of Sumo modification system:The expression vectors pCDF-Sae2-Sae1and pACYC-Ubc9-Sumo1were transfected into the E.coli to form the Sumo modificationsystem. First, the vector pACYC-Ubc9-Sumo1was transfected into BL21competent cell,then bacterium with pACYC-Ubc9-Sumo1was made to the competent cell by the Cacl2competent cell comparing methord. Finally, the plasmid pCDF-Sae2-Sae1was transfectedinto this bacterium to form the modification system.（4）The function checking of Sumo modification system:The Sumo modification ofsubstrate Oct4.The modification of susbstrate of Oct4:The vector pGEX-Oct4^k118RandpGEX-Oct4was transfected into Sumo system respectively to form the co-expressionbacterium with pGEX-Oct4/pGEX-Oct4^k118R、pCDF-Sae2-Sae1、pACYC-Ubc9-Sumo1threeplasmids. It has already detected the Sumo-Oct4protein by induction of bacterium andincubated by Oct4antibody.（5）The genetic stability detection of Sumo modification system:The bacterium withpGEX-Oct4、 pCDF-Sae2-Sae1、 pACYC-Ubc9-Sumo1three plasmids was passagedcontinously.Each generation was extracted the plasmids to detected whether the three plasmidexist meanwhile by PCR using specific primers Sumo1、Sae2、Oct4. When passaged15generations, the object fragments were also detected. It is proved the stability of system iswell.