The application of high-performance liquid chromatography for the resolution of proteins encoded by the human adenovirus type 2 cell transformation region

Abstract

The human adenovirus 2 (Ad2) transformation genes are located in early region E1a (map position (mp) 1.3–4.5) and E1b (mp 4.6–11.2) on the linear duplex Ad2 DNA genome of M r 23 × 10 6 (viral DNA is divided into 100 map units). E1b codes for three major proteins of apparent molecular weights 53,000 (53K), 19K, and 20K; smaller quantities of 21K, 22K, and 23K proteins that are related to 53K are also synthesized in Ad2-infected cells. Because the resolution and purification of these Ad2 candidate transformation proteins proved very difficult by conventional protein purification methods, the applicability of high-performance liquid chromatography (HPLC) methodology was examined. Starting with a crude cytoplasmic S100 fraction of Ad2-infected human cells, the resolution of the Ad2 E1b-coded 19K, 20K, 21K, 22K, and 23K proteins by reverse-phase HPLC using a C 8 column and a linear 0–60% 1-propanol gradient in 0.5 m pyridine formate was achieved, E1b proteins purified under these conditions retained their immunological reactivity. By anion-exchange HPLC using a linear 10 m m to 1 m NaCl gradient in 10 m m 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.6, the same five Ad2 E1b-coded 19K–23K proteins were separated, with improved resolution of the 19K protein. Based on these findings, protocols for the extensive purification of the E1b-19K and E1b-20K proteins have been developed. These results illustrate the potential of HPLC methodology for the rapid purification of biologically interesting proteins from complex cellular mixtures of proteins.