Bacterial artificial chromosomes improve recombinant protein production in mammalian cells.
BACKGROUND
The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production.


RESULTS
In this work, we explore the efficacy of a Bacterial Artificial Chromosome based vector applied to production of the constant region of the human IgG1. Direct comparison of bulk HEK 293 cell cultures generated with a "conventional" vector or with a BAC-based vector showed that the BAC-based vector improved the protein yield by a factor of 10. Further analysis of stable cell clones harboring the BAC-based vector showed that the protein production was directly proportional to the number of integrated BAC copies and that the protein production was stable for at least 30 passages.


CONCLUSIONS
Generation of stable cell clones for protein production using Bacterial Artificial Chromosomes offers a clear advantage over the use of conventional vectors. First, protein production is increased by a factor of 10; second, protein production is stable overtime and third, generation of BAC-based expression vectors does not imply a significant amount of work compare to a conventional vector. Therefore, BAC-based vectors may become an attractive tool for protein production.
Blaas
Leander
L
Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Währinger Str, 13a, A-1090 Vienna, Austria. Leander.Blaas@lbicr.lbg.ac.at
Musteanu
Monica
M
Eferl
Robert
R
Bauer
Anton
A
Casanova
Emilio
E
D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*
D007074:Immunoglobulin G; D011994:Recombinant Proteins