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 expressed in malignancies with latency type II and III and is an important transforming protein. To further study this protein LMP1 was expressed by and purified from recombinant baculovirus infected Sf9 cells. Expression levels of LMP1 in EBV transformed B cell lines and Sf9 cells were analyzed using a newly developed quantitative LMP1-capture ELISA. Highest expression was found 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. Surprisingly bLMP1 could also be detected in the culture medium as a stable full-length protein. Highest expression in Sf9 cells (506 ng/10(7) cells) was observed at 48 h post infection and in the culture medium (1590 ng/ml) at 96 h post infection. Before purification bLMP1 was solubilised using 0.22 m octyl-beta-glucoside at pH 6.0. Purification of bLMP1 using Q-Sepharose FF yielded 10-80 times enriched bLMP1 fractions, indicating that Q-Sepharose can be used for pre-purification. A one-step monoclonal antibody based immunoaffinity chromatography yielded highly purified bLMP1. Although the overall yields (20 microg purified LMP1 from 100 ml culture supernatant) and protein concentrations were low, higher concentrations of >95% purified BLMP1 could be reached after freeze drying.
Meij
P
P
Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands. p.meij@azvu.nl
Vervoort
M B
MB
Meijer
C J
CJ
Bloemena
E
E
Middeldorp
J M
JM
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D016367:Baculoviridae / Q000235:genetics; D002144:Callithrix; D002460:Cell Line; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007313:Insecta; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D012685:Sepharose