Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.
Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.
Hrzenjak
A
A
Institute of Medical Biochemistry and Medical Molecular Biology, University of Graz, Austria.
Frank
S
S
Maderegger
B
B
Sterk
H
H
Kostner
G M
GM
D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D007457:Iodine Radioisotopes; D018082:Kringles / Q000235:genetics*; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose
D001054:Apolipoproteins A; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D006639:Histidine; D012685:Sepharose; D008239:Lysine