Preparation of the trimeric subunits of recombinant human mannan-binding lectin and analysis of its bioactivity / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities.</p><p><b>METHODS</b>A prokaryotic expression vector containing human MBL N-terminal deletant (rhMBLδN) gene we previously constructed was transformed into E. coli for efficient expression of rhMBLδN fusion protein. Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins, rhMBLδN fusion protein was limitedly hydrolyzed with thrombin. The obtained rhMBLδN polypeptide was repeatedly dialyzed in 50 mmol/L PBS (pH7.2) and ddH(2)O, and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay.</p><p><b>RESULTS</b>rhMBLδN polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBLδN fusion protein with thrombin. Repeated dialyses of rhMBLδN polypeptides in 50 mmol/L PBS and ddH(2)O resulted in the isolation of the trimeric subunit trhMBLδN (with a relative molecular mass of about 50 000), which contained a collagen-like helix. The trhMBLδN protein had a higher ligand-binding activity than rhMBLδN polypeptide, and acquired the activity to initiate the lectin pathway of complement activation, but the activities were lower than those of natural MBL.</p><p><b>CONCLUSION</b>We have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.</p>