Abstract: ObjectiveTo prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological
activities. MethodsA prokaryotic expression vector containing human MBL N-terminal deletant (rhMBL△N) gene we
previously constructed was transformed intoE. colifor efficient expression of rhMBL△N 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, rhMBL△N fusion protein was limitedly hydrolyzed with
thrombin. The obtained rhMBL△N polypeptide was repeatedly dialyzed in50mmol/L PBS (pH7.2) and ddH2O, and the final
product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay.ResultsrhMBL△N
polypeptide with a relative molecular mass of about20000was obtained by limited proteolysis of rhMBL△N fusion protein
with thrombin. Repeated dialyses of rhMBL△N polypeptides in50mmol/L PBS and ddH2O resulted in the isolation of the
trimeric subunit trhMBL△N (with a relative molecular mass of about50000), which contained a collagen-like helix. The
trhMBL△N protein had a higher ligand-binding activity than rhMBL△N polypeptide, and acquired the activity to initiate the
lectin pathway of complement activation, but the activities were lower than those of natural MBL. ConclusionWe have
successfully obtained the bioactive trimeric subunit of rhMBL, trhMBL△N, and this structural subunit is also the functional
subunit of the MBL molecule.