Expression of Human COL1A1 Gene in Stably Transfected HT1080 Cells: The Production of a Thermostable Homotrimer of Type I Collagen in a Recombinant System

Abstract

A recombinant system was developed for the production of homotrimeric type I collagen in stably transfected HT1080 cells. A DNA construct (COL1A1-CMV) was prepared that contained the cDNA for the human COL1A1 gene under the transcriptional control of the promoter and enhancer of the immediate early gene of CMV. The construct, which also contained a neomycin-resistance gene, was transfected into HT1080 cells, a human fibrosarcoma cell line that synthesizes type IV collagen but does not normally synthesize any of the fibrillar collagens. Cells derived from the neomycin-resistant tranfectants were then screened using a polyclonal antibody specific for human proal (I) chains in order to identify clones that secreted high levels of the proa(I) chain of type I procollagen. About 2% of neomycin-resistant clones secreted procollagen that consisted of a homotrimer of proα1(1) chains. The procollagen was post-translationally over-modified as judged by slower migration on SDS-polyacrylamide gel electrophoresis of the proα1(1) chains compared to proα1(1) chains of normal type I procollagen. The procollagen was triple helical as assayed by protease digestion with a variable cleavage at 38 °C and a thermal transition of both the intact and partially cleaved protein of about 41 °C. The system provides a method of expressing genes for fibrillar procollagens so that fully recombinant proteins are generated and easily isolated.