Fast and high yield recombinant proteins production by large-scale transfection of suspension-growing mammalian cells, L-11266
High yield and low cost protein production in a few days to a couple of weeks (mg or g of protein). Production of proteins, including antibodies, for High-throughput screening assays, antibody generation and structure-activity analyses.
Full description
SUMMARY:
Mammalian expression systems allow for the production of
active recombinant proteins (r-proteins) that possess relevant
post-translational modifications. Current practices for the
production of significant amounts of r-proteins with
mammalian cells are often long, tedious, or of limited
scalability. This technology provides an improved process for
the production of r-proteins by the transient transfection of
suspension-growing cells. The process carried out in a single
step is easily scalable and achieves high expression levels in
a very short period of time.
APPLICATIONS:
High yield and low cost protein production in a few
days to a couple of weeks (mg or g of protein).
Production of proteins, including antibodies, for
High-throughput screening assays, antibody generation,
structure-activity analyses, surface plasmon resonance, NMR
or crystallography.
CONCEPT:
Transient gene expression is often preferable to the
establishment of stable transfectant as this latter approach is
time consuming and requires that the expressed protein not
adversely affect the growth of the cells. This technology
provides a robust large-scale transient transfection process
for fast production of milligram to gram amounts of r-proteins
in suspension-growing mammalian cells. This easily scalable
technology, amenable to high throughput production was
obtained by combining optimized parameters in four key
aspects namely the cell line, the expression vector, the
transfection vehicle and the culture medium. The vectors,
medium and cell line were optimized to allow highest
expression levels in a short period of time. In addition,
recovery of secreted r-proteins is easily achieved as the
process uses serum-free medium.
FEATURES AND BENEFITS:
Optimized serum-free adapted cell line -
A novel cell line, designated 293SFE, was established by
stably transfecting serum-free adapted HEK293SF-3F6 cells
with an EBNA1 (Epstein-Barr virus (EBV) Nuclear Antigen 1)
expression plasmid. Although the commercially available
HEK293E cell line also allows the episomal persistence of
vectors containing the EBV origin of replication oriP, the
293SFE cell line offers the added advantage of being capable
of growing in a serum-free medium.
Highly efficient expression vector -
Transient protein expression is achieved using the small size
pTT5 vector (4.4 kb), a family of expression vectors that
contains the EBV oriP and an improved cytomegalovirus-
based expression cassette. As illustrated, use of the pTT
vector in HEK293E cells provided a 2-3 fold increase in
transgene expression compared with the pCEP4 vector and a
10-fold increase compared to pcDNA3.1 vector (both from
Invitrogen).
Fast and scalable transfection -
Use of commercially-available transfection reagents led to the
development of a robust transient transfection procedure that
can be carried out at multi-liters scale. The single step
procedure is fast and easy to perform as there is no need to
change the culture medium.
High yield and cost-effective serum-free culture -
Optimization of the culture conditions led to the formulation of
a serum-free growth medium enriched with selected
peptones. This medium reduces the costs associated with r-
protein purification and allows higher transient gene
expression in the absence of serum. Protein expression
levels obtained with 293SFE cells grown in the optimized
serum-free medium were nearly identical to those obtained
with HEK293E cells cultured in a serum-supplemented
medium.
Validated expression system -
The resulting single step high performance system was
validated by the successful production and purification (>
95% purity was achieved following a single IMAC purification
step) of over thirty r-proteins; scale ranged from 1 to 14-liters
while expression yields ranged from 20 to 60 mg/L for non-
toxic proteins.
Patent information
Enhanced production of recombinant proteins by transient
transfection of suspension-growing mammalian cells (NRC
no. 11266).
Canada, Application number 2446185 , Filed 2002-05-07.
European Common Market, Application number 20.727108,
Filed 2002-05-07.
Singapore, Application number 200306591-9, Filed 2002-05-
07.
United States, Application number 10/477148, Filed 2003-11-
07.
Type of business relationship sought
Non exclusive, for R&D and commercial use.