Page 27 - Macromolecular Crystallography

16 MACROMOLECULAR CRYS TALLOGRAPHY

stocks and limitation of expression for a short time DNA is mixed with calcium phosphate to form a
due to cytopathic effects of viral infection. fine precipitate which is dispersed in the cultured
In the case of recombinant protein production for cells; and (2) DEAE-Dextran mediated transfec-
structural biology applications and specifically for tion (McCutchan and Pagano, 1968) where DNA
obtaining milligram quantities of protein for crys- is mixed with DEAE-Dextran and dispersed in the
tallization, direct transfection of vector DNA is a cultured cells. An alternative method is cationic
convenient option mainly due to advances in trans- lipid mediated transfection (Felgner et al., 1987).
fectionmethodsandreagents. Vectorscontainingthe Cationic headgroups of the lipid associate with the
gene of interest require the following essential fea- negatively charged phosphates on the DNA. The
tures: (1) a bacterial origin of replication for vector lipid-DNA complexes contact the cell membrane
propagation in E. coli; (2) a constitutive or inducible and fusion results in the internalization of DNA
promoter; (3) mRNA cleavage and polyadenylation into the cell. This is a highly efficient, reproducible
signal; (4) a transcription termination signal; (5) a method of transfection with low toxicity that is
Kozak sequence for optimal ribosome binding; (6) ideal for large scale protein expression. There are
a translation termination signal; and (7) in the case a number of commercially available, lipid-based
of transient expression an SV40 (or other viral) ori- transfection reagents but a much cheaper alterna-
gin of replication for maintenance of vector DNA tive for large-scale transient transfections is to use
in host cell. Additional features such as purification polyethylenimine (PEI), an organic macromolecule
tags, secretion signals, fusion moieties, and protease which is low in toxicity and yields high transfection
cleavage sites can also be engineered. efficiencies.
Vectors containing the target gene in an expression Proteins can be expressed intracellularly or
cassette are engineered with the Kozak sequence secreted into the growth medium. For example
and, if desired, an appropriate purification tag if one aims to express the extracellular domain
downstream of a powerful promoter. Strong viral of a cell surface receptor, the engineered gene
promoters from cytomegalovirus (CMV) or SV40 sequence can be cloned into the expression vec-
are commonly used in most mammalian expression tor with the native membrane targeting signal.
vectors. The elongation factor (EF)-1 promoter is a Providing that the signal sequence is recognized
widely used non-viral promoter due to equivalent in mammalian cells, the recombinant gene prod-
or even better expression levels compared to viral uct will be secreted in into the medium. Like-
promoters. wise, N-terminal secretion signal sequences from
Human embryonic kidney (HEK) 293, baby ham- other proteins can be engineered with gene or gene
ster kidney (BHK), and COS cells are commonly fragments of interest to secrete a protein or its
used in transient expression systems due to the high subdomains.
transfection efficiencies with these cell lines. Genet- After the uptake of foreign DNA, extrachromo-
ically modified HEK-293 and COS lines that express somal replication peaks at around 48 h post trans-
the SV40 large T antigen (HEK 293T, COS-1, -3, fection after which cells begin to shed the high
and -7) or the Epstein–Barr Virus nuclear antigen number of plasmid copies. This is followed by
(HEK 293 EBNA) are particularly preferred. Plas- cell death, probably due to the inability to endure
mids carrying SV40 or EBV origins of replication the presence of excessive quantities of extrachro-
are amplified and maintained by high extrachromo- mosomally replicating DNA (Gerard and Gluz-
somal replication levels when used with these cell man, 1985; Geisse, et al., 1996). In the case of
lines. Thus when used in combination with a pow- COS cells, recombinant protein expression peaks at
erful eukaryotic or viral promoter (e.g. SV40, CMV around 72 h after transfection. However, in spite
or EF-1) high transcription and translation levels of cell deterioration and death, expression contin-
of target genes can be achieved. Traditional chem- ues for a further 5–10 days (Edwards and Aruffo,
ical methods of introducing DNA into eukaryotic 1993). Typically, with COS and HEK 293T cells
cell cultures are: (1) Calcium phosphate mediated protein expression can be allowed for 3–4 days
transfection (Graham and van der Eb, 1973), where post-transfection.