title: Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.
abstract: In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' 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 overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'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 α'E dose (2 μM) were similar to those found in cells incubated with 1 μM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'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.
authors.affiliation: Department of AgriFood Molecular Sciences, Università degli Studi di Milano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Consonni
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Lovati
authors.forename: Anna
authors.initials: A
authors.lastname: Parolari
authors.forename: Cristina
authors.initials: C
authors.lastname: Manzoni
authors.forename: Paolo
authors.initials: P
authors.lastname: Morazzoni
authors.forename: Chiara
authors.initials: C
authors.lastname: Magni
authors.forename: Marcello
authors.initials: M
authors.lastname: Duranti
mesh_terms: D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005916:Globulins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D050356:Lipid Metabolism / Q000187:drug effects; D008077:Lipoproteins, LDL / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D055314:Seed Storage Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D030262:Soybean Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D013025:Glycine max / Q000235:genetics*
chemical_list: D052179:Antigens, Plant; D005916:Globulins; D008077:Lipoproteins, LDL; D021122:Protein Subunits; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; D002784:Cholesterol