Prokaryotic Expression Plasmid Construction, Expression, Purification And Function Detection Of Penetratin/NF-κB Antagonizing Polypeptide

NF-κB is a ubiquitous transcription factor, and it plays a key role in basic processes such as regulation of the immune and inflammatory responses, virus replication, cell proliferation and apoptosis. It can effectively induce the transcription and expression of more than 60 genes such as cytokines, adhesion molecules, chemotaxins, acute phase response proteins, and also regulate the expression of many enzymes, which take part in inflammatory responses. Over expression of NF-κB is associated with pathogenesis and processes of various diseases, such as cancer, autoimmune disease, chronic inflammatory diseases, allergy, and arteriosclerosis, graft rejection. Therefore, many experts suggested recently that NF-κB could be a potential novel anti-inflammatory target. Theoretically, effectively antagonizing NF-κB activity is one of the effective ways to relieve and/or treat those inflammatory diseases.NF-κB could bind to DNA cis-element specifically, Based on this, DNA-binding domain of p65 subunit and Rel homology domain (RHD) of p50 were used as"prayers", via the yeast two-hybrid system from a random 16-peptides cDNA library, Our team have screened 14 polypeptides. Among these polypeptide, 5 interact with DNA-binding domain of p65 subunit and 9 with another subunit's RHD. But, some false positive maybe exist in the yeast two-hybrid technology. So, additional methods must be done to confirm the result. Additionally, through the sequencing of these polypeptides, we found that these polypeptides are hydrophobic ones. Direct manual synthesis of these polypeptides is water insoluble. To get soluble and specific NF-κB interacting polypeptide, we need to recombinant these cDNA to a prokaryotive expression plasmid and express them via Escharichia coli strain BL-21(DE3) and purification was done subsequently.To obtain purified polypeptide, the following experiments were performed in this study.1.Construction of pET42a/P/GST fusion plasmidPlasmid pET42a were cut by NcoⅠand BamHⅠseparately. After gel extract purification, pET42a fragment were mixed up with peptide cDNA fragment, which was synthesized directly. Then restriction endonuclease analyze had done to check whether right ligation. After this, sequencing carries out shows that open reading frame of target fragment is right. Plasmid constructed successfully and was named pET42a/PGST.2.Construction of pET42a /PP/GST fusion expression plasmid Amplified by PCR method, the cDNA of polypeptide were cloned in frame with glutathione S-transferase (GST) tag open reading frame of the plasmid constructed above. Then identified by PCR and restriction endonuclease analyze. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct. And it was named pET42a /PP/GST.3.Expression of GST-polypeptide fusion proteins in Escharichia coli strain BL-21.The recombinant plasmid were transformed into E.coli BL-21 (DE3) and induced in 0.25mmol/L isopropyl-1-thio-β-D-galactopyranoside(IPTG) at 32℃for 5hr.SDS-PAGE revealed that GST-polypeptide fusion protein bands located in position of 31 kDu.Their expression level can reach to 28% of total protein. At this condition, a large scale of GST-polypeptide fusion protein was obtained, and was purified using his-tag affinity technology.4.Purification and GST tag splict of fusion proteinAfter supernatant elicited from nicel column with gravity, most target protein is combined onto nicel bead. Wash three times with wash buffer to wash off non-specific binding. Then wash several times with Xa factor solution to splict GST tag and collect target protein.5.Activity detection of penetratin/peptide.We send purification solution to SBS Co., LTD (Beijing) to C termination fluorescence labeling. And the labeled product was resolved in sterilized water. Add target peptide solution to the broth to culture U973 and HEK293 cell. After a while, we find that penetratin/peptide can penetrate cell membrane and inter into cell plasma. This result shows that protein we obtained is active.