Bacterial Expression and Characterization of Human Recombinant Apolipoprotein(a) Kringle IV Type 9

Chung, Fung Lung; Wu, Lan-Hsin; Lee, Helen T.; Mueller, W. Thomas; Spahr, Mark A.; Eaton, Scott R.; Tian, Ye; Settimi, Philip D.; Oxender, Dale L.; Ramharack, Randy

doi:10.1006/prep.1998.0898

Protein Expression and Purification

1998

DOI: 10.1006/prep.1998.0898

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Bacterial Expression and Characterization of Human Recombinant Apolipoprotein(a) Kringle IV Type 9

Fung Lung Chung¹,

Lan-Hsin Wu²,

Helen T. Lee³

et al.

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2000

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

Hrzenjak

Frank²,

Maderegger³

et al. 2000

Protein Engineering, Design and Selection

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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.

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

Hrzenjak

Frank²,

Maderegger³

et al. 2000

Protein Engineering, Design and Selection

View full text Add to dashboard Cite

show abstract

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Bacterial Expression and Characterization of Human Recombinant Apolipoprotein(a) Kringle IV Type 9

Cited by 1 publication

References 17 publications

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

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

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