Expression of human. alpha. sub 2 -macroglobulin cDNA in baby hamster kidney fibroblasts: Secretion of high levels of active. alpha. sub 2 -macroglobulin

Journal Article · Tue May 01 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00469a009· OSTI ID:6172379

Boel, E; Mortensen, S B ^[1]; Kristensen, T; Sottrup-Jensen, L ^[2]; Petersen, C M ^[3]

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1. Novo Nordisk A/S, Bagsvaerd (Denmark)
2. Univ. of Aarhus (Denmark)
3. Skejby Sygehus, Aarhus (Denmark)

Human {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase {alpha}{sub 2}M entraps the proteinase molecule in a reaction that involves large conformational changes in {alpha}{sub 2}M. The authors describe the molecular cloning of {alpha}{sub 2}M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human {alpha}{sub 2}M with a specific enzyme-linked immunosorbent assay. Human recombinant {alpha}{sub 2}M (r{alpha}{sub 2}M), secreted and purified form isolated transfected BHK cell lines, was structurally and functionally compared to {alpha}{sub 2}M purified from human serum. The results show that r{alpha}{sub 2}M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r{alpha}{sub 2}M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference.

• OSTI ID: 6172379
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:17; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CLONING
CODONS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DNA
DNA HYBRIDIZATION
DNA-CLONING
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
FIBROBLASTS
GLOBULINS
GLOBULINS-ALPHA
HAMSTERS
HYBRIDIZATION
HYDROLASES
INHIBITION
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KIDNEYS
MAMMALS
MAN
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
PRIMATES
PROTEINS
PURIFICATION
RADIOISOTOPES
RECOMBINANT DNA
RODENTS
SECRETION
SOMATIC CELLS
VERTEBRATES