Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

Abstract

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

• Authors: A Ribeiro, P Cardot, R Benarous, P Pernas, D Pepin, T Rybkine, J Chambaz, G Bereziat
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1087 Issue 2 Pg. 219-25 (Oct 23 1990) ISSN: 0006-3002 [Print] Netherlands
• PMID: 2223883 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Apolipoproteins E
• Codon
• RNA, Messenger
• Receptors, LDL
• Recombinant Fusion Proteins

Topics

• Apolipoproteins E (genetics, metabolism)
• Base Sequence
• Cells, Cultured
• Cloning, Molecular
• Codon
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (enzymology, genetics)
• Gene Expression
• Humans
• Hypercholesterolemia (metabolism)
• Molecular Sequence Data
• RNA, Messenger (genetics)
• Receptors, LDL (metabolism)
• Recombinant Fusion Proteins (genetics, metabolism)
• Restriction Mapping