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

P Meij, M B Vervoort, C J Meijer, E Bloemena, J M Middeldorp

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)193-204
Number of pages12
JournalJournal of Virological Methods
Volume90
Issue number2
Publication statusPublished - Nov 2000

Cite this

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title = "Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected insect cells",
abstract = "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.",
keywords = "Animals, Antibodies, Monoclonal, Antibodies, Viral, Baculoviridae, Callithrix, Cell Line, Cell Line, Transformed, Chromatography, Affinity, Enzyme-Linked Immunosorbent Assay, Insecta, Oncogene Proteins, Viral, Recombinant Proteins, Sepharose, Transfection, Viral Matrix Proteins, Comparative Study, Journal Article",
author = "P Meij and Vervoort, {M B} and Meijer, {C J} and E Bloemena and Middeldorp, {J M}",
year = "2000",
month = "11",
language = "English",
volume = "90",
pages = "193--204",
journal = "Journal of Virological Methods",
issn = "0166-0934",
publisher = "Elsevier",
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}

Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected insect cells. / Meij, P; Vervoort, M B; Meijer, C J; Bloemena, E; Middeldorp, J M.

In: Journal of Virological Methods, Vol. 90, No. 2, 11.2000, p. 193-204.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected insect cells

AU - Meij, P

AU - Vervoort, M B

AU - Meijer, C J

AU - Bloemena, E

AU - Middeldorp, J M

PY - 2000/11

Y1 - 2000/11

N2 - 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.

AB - 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.

KW - Animals

KW - Antibodies, Monoclonal

KW - Antibodies, Viral

KW - Baculoviridae

KW - Callithrix

KW - Cell Line

KW - Cell Line, Transformed

KW - Chromatography, Affinity

KW - Enzyme-Linked Immunosorbent Assay

KW - Insecta

KW - Oncogene Proteins, Viral

KW - Recombinant Proteins

KW - Sepharose

KW - Transfection

KW - Viral Matrix Proteins

KW - Comparative Study

KW - Journal Article

M3 - Article

VL - 90

SP - 193

EP - 204

JO - Journal of Virological Methods

JF - Journal of Virological Methods

SN - 0166-0934

IS - 2

ER -