In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin alfa' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and over-expressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed alfa'E, was 8-fold greater than the previous truncated version. The alfa'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest alfa'E dose (2 microM) were similar to those found in cells incubated with 1 microM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to alfa'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

Heterologous expression and purification of the soybean 7S globulin α′ subunit extension region: In vitro evidence of its involvement in cell cholesterol homeostasis / A. Consonni, M.R. Lovati, A. Parolari, C. Manzoni, P. Morazzoni, C. Magni, M. Duranti. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 80:1(2011), pp. 125-129.

Heterologous expression and purification of the soybean 7S globulin α′ subunit extension region: In vitro evidence of its involvement in cell cholesterol homeostasis

A. Consonni;M.R. Lovati;C. Manzoni;C. Magni;M. Duranti
2011

Abstract

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin alfa' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and over-expressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed alfa'E, was 8-fold greater than the previous truncated version. The alfa'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest alfa'E dose (2 microM) were similar to those found in cells incubated with 1 microM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to alfa'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.
7S soybean globulin ; alfa' chain ; extension region ; Hep G2 cells ; LDL-receptors ; cell cholesterol homeostasis
Settore BIO/14 - Farmacologia
Settore BIO/10 - Biochimica
2011
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/163591
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