Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.
Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.
Crucell Holland BV, PO Box 2048, 2301CA Leiden, The Netherlands. m.havenga@crucell.com
M J E
MJ
Havenga
L
L
Holterman
I
I
Melis
S
S
Smits
J
J
Kaspers
E
E
Heemskerk
R
R
van der Vlugt
M
M
Koldijk
G J
GJ
Schouten
G
G
Hateboer
K
K
Brouwer
R
R
Vogels
J
J
Goudsmit
D000256:Adenoviridae / Q000235:genetics*; D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D016895:Culture Media, Serum-Free; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins