[Prokaryotic expression of N terminal fragment of mannan-binding lectin associated serine protease-1].
OBJECTIVE
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.


CONCLUSIONS
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.
Department of Immunology, Southern Medical University, Guangzhou, China.
Xue-min
XM
Cai
Na
N
Zhao
Da-ming
DM
Zuo
Li-yun
LY
Zhang
Zheng-liang
ZL
Chen
D020816:Amino Acid Motifs; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D037601:Mannose-Binding Lectin / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins; C084067:MASP1 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases