Aim: To express the N-terminal fragment of human mannan-binding lectin (MBL) associated serine proteases-1 (MASP1-N) in E.coli.
Methods: The target sequence in pGEM-MASP1 plasmid that contains human MBL-MASP1 cDNA was amplified by PCR, inserted into prokaryotic expression vector pGEX4T-1 and identified by restriction mapping and sequencing. The recombinant expression vector was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by GSTrap Immobilized Metal Affinity Chromatography(IMAC) and identified by SDS-PAGE and Western blot assay, its binding-activity with the collagen-like region of human MBL(MBL-CLR) and with recombinant human MBL was analyzed by an indirect enzyme-linked immunosorbent assayèELISAé.
Results: The DNA fragment of 860 bp, which encode the N-terminal region of human MASP1, was amplified from pGEM-MASP1 plasmid and the recombinant expression vector, pGEX4T-MASP1-N, was constructed, whose restriction maps and sequence were consistent with those expected. The component of M(r) 60 000 in the purified recombinant product was found by SDS-PAGE and could be recognized by anti-GST antibody in Western blot assay. The purified recombinant product could react with human MBL-CLR and human MBL in the indirect ELISA.
Conclusion: The prokaryotic cell strain that expresses efficiently recombinant human MASP1-N(rhMASP1-N) protein and the purified rhMASP1-N protein were successfully obtained, which provides the basis for further research of MASP1 molecule.