Expression of a recombinant kringle V of human apolipoprotein(a): antibody characterization and species specificity

Protein Expr Purif. 1996 Sep;8(2):145-50. doi: 10.1006/prep.1996.0086.

Abstract

Lipoprotein(a) is a macromolecular complex consisting of a low-density lipoprotein-like particle with an additional glycoprotein, apolipoprotein(a) [apo(a)], linked to apolipoprotein B-100 via a disulfide bond. Apo(a) is highly homologous to plasminogen. We have cloned the sequence corresponding to the kringle V domain of apo(a) from human liver cDNA using an experimental approach involving use of the polymerase chain reaction. The protein product of this clone was expressed in the cytoplasmic compartment of Escherichia coli as a MalE fusion protein. Fusion apo(a) Kr V was isolated from cytoplasmic extracts and purified by amylose-agarose affinity chromatography by eluting with 10 mM maltose. The fusion protein was injected into sheep in order to generate a polyclonal anti-apo(a) Kr V antibody. The antibody raised reacted against both reduced Lp(a) and the C-terminal domain of apo(a), corresponding to a sequence extending from Kr 33 to the C-terminal residue, but did not react with the N-terminal domain containing the repeated Kr IV sequences. The presence of the Kr V sequence was detected in every human apo(a) size isoform tested but only in apo(a) from human and chimpanzee among a panel of apo(a) proteins derived from different animal species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters*
  • Antibodies / immunology*
  • Antibodies / metabolism
  • Apolipoproteins A / genetics*
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli Proteins*
  • Gene Expression / genetics
  • Humans
  • Kinetics
  • Kringles / genetics*
  • Liver / metabolism
  • Maltose-Binding Proteins
  • Monosaccharide Transport Proteins*
  • Periplasmic Binding Proteins*
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Species Specificity*

Substances

  • ATP-Binding Cassette Transporters
  • Antibodies
  • Apolipoproteins A
  • Carrier Proteins
  • DNA Primers
  • Escherichia coli Proteins
  • MalE protein, E coli
  • Maltose-Binding Proteins
  • Monosaccharide Transport Proteins
  • Periplasmic Binding Proteins
  • Recombinant Fusion Proteins
  • maltose transport system, E coli