An efficient expression vector for extracellular secretion in mammalian cells

Abstract

An expression-secretion vector for mammalian cells, pcDSA, which expresses a cloned gene under the control of the SRa promoter (SV40 promoter/enhancer and HTLV-1 LTR) has been newly constructed. This vector contains fragments encoding the 5′ untranslated leader sequence from AMV RNA4, the signal peptide of mouse IgM and IgG-binding domain of protein A in front of cloning sites. Joining in-frame a cDNA fragment with cloning sites just downstream of the COOH terminus of the IgG-binding domain of protein A enables the cDNA product to be secreted as a protein fused with that domain. This allows an easy isolation of its secreted product by affinity chromatography on IgG-Sepharose. When the genes encoding the catalytic domains of mammalian sialyltransferase (ST3Gal I) were cloned into the vector plasmid and then transfected into COS-7 cells, active ST3Gal I was efficiently secreted into the culture medium. It warf rapidly purified almost to homogeneity by one-step IgG-Sepharose affinity chromatography.