Cloning, yeast expression, purification and biological activity of a truncated form of the soybean 7S globulin α′ subunit involved in Hep G2 cell cholesterol homeostasis☆

Abstract 

A truncated form of α′ chain (tα′), the soybean 7S globulin subunit previously demonstrated to be active in controlling the cholesterol and triglyceride homeostasis in in vitro and in vivo models, was cloned and expressed in the yeast Pichia pastoris. The recombinant polypeptide spanned 216 amino acid residues from the N-terminal side and included the N-terminal extension region of the soybean subunit. The tα′ polypeptide was purified by conventional biochemical techniques, and its potential to modulate the activity of low-density lipoprotein (LDL) receptor was evaluated in a human hepatoma cell line (Hep G2) by monitoring the uptake and degradation of labeled LDL. The LDL uptake (+192%) and degradation (+143%) by cells tested at the highest tα′ dose (8 μM) were similar to those found in cells incubated with 1 μM simvastatin, a potent inhibitor of cholesterol biosynthesis. The cell response to tα′ was found to be dose dependent. The use of a recombinant polypeptide ruled out the involvement of any other soybean component.

These findings open new prospects in the studies aimed at identifying soybean regulatory (poly)peptide(s) and the mechanism involved in this biological response, as a gateway to their utilization for the management of human health.

Abbreviation: FCS, fetal calf serum, LDH, lactate dehydrogenase, LDL-R, LDL receptor, LPDS, lipoprotein-deficient serum, MEM, minimum essential medium, MTT, methyltetrazolium salt, PCR, polymerase chain reaction, tα′, truncated form of 7S soybean globulin α′ subunit

Keywords: 7S Soybean proteins, Truncated α′, Chain, Hep G2 cells, LDL Receptors, Cell cholesterol homeostasis