title: [Preparation of the trimeric subunits of recombinant human mannan-binding lectin and analysis of its bioactivity].
abstract: OBJECTIVE
  To prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  We have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.
authors.affiliation: Department of Immunology, Southern Medical University, Guangzhou, China. pual-lei@hotmail.com
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.forename: Junrong
authors.initials: J
authors.lastname: Tong
authors.forename: Xiao
authors.initials: X
authors.lastname: Lu
authors.forename: Liyun
authors.initials: L
authors.lastname: Zhang
authors.forename: Daming
authors.initials: D
authors.lastname: Zuo
authors.forename: Zhengliang
authors.initials: Z
authors.lastname: Chen
mesh_terms: D003167:Complement Activation; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D037601:Mannose-Binding Lectin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins