RESUMO
Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.
RESUMO
Objective:To express and purificate catalytic domain of murine?-1,3-galactosyltransferase and provide the feasible method in the tumor cell surface production ?-gal epitopes..Methods:This research established expression system in pET-15b to express catalytic domain of murine ?-1,3-galactosyltransferase,then identified its activity by HPLC with anion exchange column.Results:(1)Constructed successfully recombinant ?-1,3-galactosyltransferase catalytic domain with His-tag.(2)?-1,3-galactosyltransferase with His-tag in a soluble form was expressed and purificated effeciently.(3)Its activity by HPLC with anion exchange column showed.Conclusion:This research shows ?-1,3-galactosyltransferase in a soluble form has been expressed successfully.