Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.
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.
Ribeiro
A
A
UPA CNRS 1283, CHU Saint-Antoine, Paris, France.
Cardot
P
P
Benarous
R
R
Pernas
P
P
Pepin
D
D
Rybkine
T
T
Chambaz
J
J
Bereziat
G
G
D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006937:Hypercholesterolemia / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011973:Receptors, LDL / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D001057:Apolipoproteins E; D003062:Codon; D012333:RNA, Messenger; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins