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Selected Publication:
Hrzenjak, A; Frank, S; Maderegger, B; Sterk, H; Kostner, GM.
Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.
Protein Eng. 2000; 13(9):661-666
Doi: 10.1093%2Fprotein%2F13.9.661
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- Leading authors Med Uni Graz
- Hrzenjak Andelko
- Kostner Gerhard
- Co-authors Med Uni Graz
- Frank Sasa
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- Abstract:
- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Apolipoproteins A - chemistry
- Base Sequence - chemistry
- Cloning, Molecular - chemistry
- Escherichia coli - genetics
- Histidine - genetics
- Iodine Radioisotopes - genetics
- Kringles - genetics
- Lysine - genetics
- Magnetic Resonance Spectroscopy - genetics
- Molecular Sequence Data - genetics
- Polymerase Chain Reaction - genetics
- Protein Engineering - methods
- Protein Folding - methods
- Recombinant Proteins - chemistry
- Sepharose - chemistry
- Find related publications in this database (Keywords)
- apo(a)
- H-1-NMR
- kringle
- Lys-Sepharose
- refolding