Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding

• Published in: Protein engineering Vol. 13; no. 9; pp. 661 - 666
• Main Authors: Hrzenjak, A, Frank, S, Maderegger, B, Sterk, H, Kostner, G M
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.09.2000
• Subjects: Apolipoproteins A - chemistry; Apolipoproteins A - genetics; Apolipoproteins A - isolation & purification; Apolipoproteins A - metabolism; Base Sequence; Cloning, Molecular; Escherichia coli - genetics; Histidine - genetics; Iodine Radioisotopes; Kringles - genetics; Lysine; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Polymerase Chain Reaction; Protein Engineering - methods; Protein Folding; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sepharose
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/13.9.661

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.