Using the bacterial artificial chromosomes for improving recombinant protein production in mammalian cells

In recent years, global demand for protein has continued to grow at an unprecedented rate and the process will continue. For example, the market for monoclonal antibodies is predicted to rise hugely in the next few years, and the demand for many antibodies is expected to be in excess of 100 kg per years (1). Because users need quality protein at a good price, the research must continue to refine the process of biosynthesis. Today, the mammalian cells are the dominant system for the production of recombinant proteins for clinical applications because the tertiary structure of proteins and their glycosilation are similar to human proteins (2). Nevertheless, the use of mammalian cells raises some problems of productivity, such as DNA delivery and integration, host cell engineering, medium optimization, selection of the gene(s) amplification and cell line screening (2). In this context, in the scientific article ‘‘Bacterial artificial chromosome improve recombinant protein production in mammalian cells’’ Blaas, L. et al. say that using a Bacterial Artificial Chromosome (BACs), instead of a ‘‘classical vector expression’’, increases the efficiency of biosynthesis.

The researchers, analyzing the processes used to obtain and cultivate transgenic cell lines, have observed that the integration of genes into the host chromosome is important for the rate of transcription of recombinant genes. In addition, they indicated that this random process could be masked by the position effect (3). Wurm, F., 2004, said that transgene expression in mammalian cells is rapidly inactivated (silenced) in many cases, probably because of the influence of neighbouring condensed chromatin. In agreement with Wurm, F., the authors of the article pointed out that the production of the protein is not stable due the position effect. Theirs working hypothesis was that the use BACs as an expression vector could increase the production of recombinant proteins, because the genes are considered as open chromatin (highly transcribed).

In their experiment they introduced a fragment (Fc) of the constant region of human IgG1 in HEK 293 cells. To test the hypothesis they made comparison between the bulk HEK 293 cell cultures generated with a “conventional” vector (bulk) or with a BAC-based vector.

The steps of the synthesis
a) Creation of the BAC: the fragment (Fc) was introduced into a vector between two att and φC31 sites then the Fc vector was recombined into a BAC containing the Rosa26 locus using φC31 mediated cassette exchange into the exon 2 of the Rosa26 antisense transcript.
b) Integration in the host cells: Rosa26 - Fc -BAC vectors were linearized with NotI and transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen). The selection was done using Geneticin (G418) an aminoglycoside antibiotic similar in structure to gentamicine B1.
c) Cell culture: the cultures were grown in the absence of G418.
d) Human IgG1-Fc protein determination: using Elisa assay with - goat anti-human-IgG, Protein A – HRP and TMB substrate solution. The ELISA was measured at 450 nm with the reference wavelength 630 nm.
e) Rosa26 BAC - Fc copy number analysis: by RT-PCR with primers designated for Rosa F 5’ and Rosa R 3’.

The results demonstrated
1) BAC-based vector improves the protein production substantially: 0.5 and 5.7 pg/cell/day in the Fc classic vector and the Rosa26 BAC – Fc vector cultures, respectively,

2) Analysing the correlation between the number of transgene copies and the Fc protein yield, they suggested that the protein production is proportional to the number of integrated transgene copies when using a BAC-based expression vector,

3) Investigating long term protein production (30 passages), they observed that the yield of the Fc protein was not significantly decreasing from passage 1 to passage 30. Thus, BAC-based vectors provide stable long-term production of recombinant proteins.

Considering all this information, the authors concluded that BAC-based expression vectors represent an important tool to improve recombinant protein production.

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