Eukaryotic And Prokaryotic Expression Domain Based Mutant Proteins Of UHRF2

Objective：To construct eukaryotically expressing constructs ofdomain deletion mutants of UHRF2and verify the recombinant proteins bywestern blot. To construct all the domain-based GST(glutathione-S-transferase) fusion mutants expressing vectors of UHRF2and purifying the recombinant products for downstream use.Methods：Domain deletion mutants of UHRF2were designed according totheir locations. Respective sequences of each deletion mutants were clonedusing PCR, in particular, sequences of PHD and YDG domain deletionmutants were amplified by overlapping PCR method, and incorporated intovector pCMV-3xFlag after double restriction enzyme digestion. Eachrecombinant insert was verified by sequencing. All the deletion mutantsconstruct were transfected into HEK293cells and recombinant proteinswere confirmed by western blot.DNA fragments of each domain of UHRF2which they span wereamplified by PCR methods. Digest the PCR products with restrictionenzymes and ligate them into pGEX-4T-1vector. Express all the mutantsby IPTG induction using transformed BL21strain of E. coli. Collect andsonicate the GST-fusion mutants containing bacteria and purify the proteinsusing Glutathione Sepharose4B. Purified proteins were subjected toSDS-PAGE followed by CBB staining or immunobloting assay.Results:All the domain deletion mutants expressing vectors of UHRF2were constructed successfully and eukaryotically expressed products wereconfirmed by immunoblotting method.All the domain-based GST fusion mutants expressing vectors ofUHRF2were constructed successfully and prokaryotically expressedproducts has been purified and confirmed.Conclusion：Domain deletion mutants could be used to study subcellular locationof UHRF2or its binding sites with another candidate protein or respectivefunction of each domain.Domain-based mutants of UHRF2could be used in GST pull-downassay to study domains of UHRF2that may be implicated in binding withits partner, which would enable further understanding of its function.