Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected insect cells

Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is expres sed in malignancies with latency type II and III and is an important transf orming protein. To further study this protein LMP1 was expressed by and pur ified from recombinant baculovirus infected Sf9 cells. Expression levels of LMP1 in EBV transformed B cell lines and Sf9 cells were analyzed using a n ewly developed quantitative LMP1-capture ELISA. Highest expression was foun d in the cell line X50/7 (6.2 ng/10(7) cells), whereas expression levels of recombinant LMP1 (bLMP1) in Sf9 cells reached 506 ng/10(7) cells. Surprisi ngly bLMP1 could also be detected in the culture medium as a stable full-le ngth protein. Highest expression in Sf9 cells (506 ng/10(7) cells) was obse rved at 48 h post infection and in the culture medium(1590 ng/ml) at 96 h p ost infection. Before purification bLMP1 was solubilised using 0.22 m octyl -beta -glucoside at pH 6.0. Purification of bLMP1 using Q-Sepharose FF yiel ded 10-80 times enriched bLMP1 fractions, indicating that Q-Sepharose can b e used for pre-purification. A one-step monoclonal antibody based immunoaff inity chromatography yielded highly purified bLMP1. Although the overall yi elds (20 mug purified LMP1 from 100 mi culture supernatant) and protein con centrations were low, higher concentrations of > 95% purified bLMP1 could b e reached after freeze drying. (C) 2000 Elsevier Science B.V. All rights re served.