Procaryotic Expression of Porcine Acid-Labile Subunit of the 150-kDa Insulin-like Growth Factor Complex

摘要

Acid-labile subunit (ALS) is a 85-kDa glycosylated plasma protein which forms a 150-kDa ternary complex with 7.5-kDa insulin-like growth factor (IGF) and 40~45-kDa IGF-binding protein-3. In a previous study, the present authors prepared a porcine ALS(pALS) expression construct by inserting a pALS coding sequence into a plasmid vector following synthesis of the sequence by reverse transcription-polymerase chain reaction (RT-PCR). The expression construct, however, was subsequently found to have a mis-sense mutation at two bases of the pALS coding sequence which is presumed to have occurred through a PCR error. In the present study, the correct coding sequence was synthesized by the site-directed mutagenesis and inserted into the pET-28a (+) plasmidexpression vector containing the His-tag sequence flanking the last codon of the insert DNA. After induction of the expression construct in E. coli BL21 (DE3) cells, the resulting presumptive recombinant peptide was purified by the Ni-affinity chromatography. Upon SDS-PAGE, the affinity-purified peptide was resolved as a single band at a 66-kDa position which is consistent with the expected molecular mass of the presumptive recombinant pALS. Collectively, results indicate that a recombinant pALS peptidewas successfully expressed and purified in the present study.