IL-10_(23-57)-PE40 Secretion of Periplasm

IL-10_ 23-57 -PE40 was cloned into the prokaryotic expresion vector containing pelB signal peptide gene,The constucted plasmid pET20b- IL-10_ 23-57 -PE40 were transformed into E.coli BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),ER2566 and E. coli K12 TB1for proposed protein expression,respectively. Through changing temperature of induction or adding 0.1mol/L glucose in LB,the target protein IL-10_ 23-57 -PE40 was directed to periplasmic space as soluble form only in E.coli BL21(DE3)pLysS and was up to 15% of the total protein. Western blot analysis showed that the fusion protein may react specifically with anti-PE40 antibody. These results maybe explain translocation across the cell membrane of E.coli has to do with the characteristcs of host strain. By rationale for plasmid stability test in E.coli BL21(DE3),Rosetta(DE3),ER2566 and E.coli K12 TB1,pET20b- IL-10_ 23-57 -PE40 was only unstable target plasmids in BL21(DE3),that maybe explain why IL-10_ 23-57 -Pe40 wasn't directed to periplasmic space in BL21(DE3). However,why the target protein didn't export to periplasm of the other host strains in which have stable recombinant plasmidWhen using SDS-PAGE,DNA Star and ANThewin protein software for analysising the mechanism of immunotoxin of pseudomonas exotoxin A,our result indicate: the leader signal is necessary,but not sufficient for export into the periplasm. Translocation across the cell membrane maybe depend on characteristic of targeting agents.