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

A partial rat apo E-β-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant λGT11 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 · 10−8 M and 4.1 · 105 sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by β-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) β-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.