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 00:00:00 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]

---

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), Vol. 29:17; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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