Prokaryotic Expression, Preparation Of Polyclonal Antibody For Human SUMO-3 Gene

In order to respond quickly and efficiently to rapid changes in both their internal and external environments, cells possess a sensitive, prompt and reversible regulatory mechanism called post-translational modification. The first example of protein acting as a post-translatinal modifier is ubiquitin, and since more 15 ubiquitin related modifiers have been characterized. Among them, Small Ubiquitin-related Modifier (SUMO) is the most studied modifier. Since its discovery less than then ten years ago, four family members for SUMO (namely SUMO-1,-2,-3, and -4) and more than 50 of its substrates have been identified. Studies on SUMO have revealed many important functions like signal transduction, transcription, protein trafficking, DNA damage repair and chromosome segregation, being controlled by this family.In this study, we choose SUMO-3 as subject, one member of SUMO second family. First, we cloned SUMO-3GG by PCR from pYFP-SUMO-3. The encoding sequences of SUMO-3GG were inserted into vectors to obtain prokaryotic expression vector pET41a (+)-SUMO-3, and eukaryotic expression vectors pCMV-HA-SUMO-3, pCMV-Myc-SUMO-3, pEGFP-C1-SUMO-3, pDsRed-C1-SUMO-3, respectively. The recombinant vector pET41a (+)-SUMO-3 was transformed into E. coli BL21 (DE3) plysS. A fusion protein about 44kD was detected by SDS-PAGE and Western blot in the induced recombinant BL21 (DE3) plysS strain. The fusion protein GST-SUMO-3 was purified by the GST chromatography column. Rabbits were immunized with the purified protein, and the antiserum was obtained, processed by ELISA, Western-blot to detect the sensitivity and specificity of its antibody. Result indicated that, prokaryotic expression vector pET41a(+)-SUMO-3 was successful. The result of ELISA is positive, and Western blot confirmed that the antiserum reacted specifically to the SUMO-3 protein.Eukaryotic expression vector pEGFP-C1-SUMO-3 was transfected into Hela cell by liposome and expressed transiently, then we observed sub-cellular localization of protein SUMO-3 by fluorescence microscope. Eukaryotic expression vectors pDsRed-C1-SUMO-3 and pEGFP-C1-p53 were transfected into Hela cell by liposome and expressed transiently, then we observed the co-localization of them by fluorescence microscope. The vectors pEGFP-C1-SUMO-3 was expressed in Hela successfully and we found that Human SUMO-3 protein was localized in the nucleus. And SUMO-3 and p53 co-localize in the nucleus.