Page 28 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 17

1.2.5.2 Stable expression from the media and full MSX inhibition in this situa-
Stable lines are produced by cloning a homogeneous tion is lethal. Therefore, MSX treatment encourages
cell population from a heterogeneous cell pool and an increase in the copy number of the GS gene when
CHO cell lines are the most commonly used type cells are cultured in the absence of glutamine, so
for stable recombinant protein expression. Features effectively coamplifying any associated genes.
of vectors used to produce stable cell lines are iden- DHFR is an enzyme in the pathway for de novo
tical to those used in transient expression with an biosynthesis of purines and pyrimidines. In the
additional feature of having a drug resistance gene. absence of DHFR (i.e. in DHFR-CHO cells), purine
Stable integration of foreign DNA in to the host and pyrimidine salvage pathways are activated.
cell genome is achieved by applying drug selection These salvage pathways can be inhibited by drugs
after transfection. The frequency of DNAintegration such as methotrexate. Drug treatment encourages an
is dependent on the cell line. A plasmid encod- increaseinthecopynumberoftheresistancegene, so
ing a drug resistance marker can be cotransfected effectively coamplifying any associated genes on the
with the plasmid containing the gene of interest. transfected vector. There is usually and broad varia-
Frequency of integration of foreign DNAby cotrans- tion of the level of expression of the gene of interest
formation depends on the cell line and efficiency which is dependent on the site of integration within
of transfection. When cotransfection is inefficient it the host chromosome.
is probably best to have both the gene of interest
and the drug selection marker on a single plasmid 1.2.5.3 Glycosylation
(Kaufmann, 1990). Proteins expressed in mammalian cells undergo
When the gene of interest and the selection marker N-linked glycosylation which may cause prob-
are on two separate plasmids they may integrate lems during crystallization, particularly if there are
in chromosomal loci with differing transcriptional multiple glycosylation sites. Heavy glycosylation
activity and, as a result, a large number of cell popu- obscures the protein surface and reduces the possi-
lations will need to be screened for expression. This bility of lattice formation mediated by the protein
can be avoided by having both the gene of interest surface during crystallization. Microheterogeneity
and resistance marker on a single plasmid vector. also prevents the formation of reproducible crystal
However, there is no firm evidence that one method contacts. There are different strategies to tackle the
is better than the other. problem of glycosylation. N-linked glycosylation
Selection markers can be bacterial genes for sites can be eliminated by site directed mutagenesis
which there is no mammalian equivalent (e.g. and this is a frequently used strategy. Also, glyco-
neomycin phosphotransferase, hygromycin phos- proteins can be treated with endoglycosidases such
photransferase). But the most common are glu- as endoglycosidase H (Endo H) and N-Glycosidase
tamine synthase (GS) and dihydrofolate reductase F (PNGase F) to cleave sugar chains or cocktails of
(DHFR) gene systems. GS Gene Expression Systems exoglycosidases to shorten glycan chains. However,
is licensed by Lonza (www.lonza.com). GS is an complete deglycosylation is sometimes difficult to
allosteric enzyme required for the production of glu- achieve and sensitivity to deglycosylases can vary
tamine from glutamate and this enzyme is inhibited between proteins.
by l-methionine sulphoximine (MSX), which is a A number of mutant CHO cell lines have been
transition state analogue of the reaction: obtained by mutagenesis and selection with lectins
(Stanley, 1981). Lec3.2.8.1 (or LecR) is a mutant CHO
Glutamate ammonia + ATP ↔ Glutamine cell line that produces truncated N-linked oligosac-
+ ADP + Pi charidesoftheendoglycosidaseH(EndoH)sensitive
Man5GlcNAc2 type (Stanley, 1989). Protein fold-
CHO cells transfected with the GS minigene do not ing proceeds normally in the ER but N-glycans
require glutamine in the growth media, provided are not processed beyond the EndoH sensitive
that sufficient glutamate is present. However, GS is Man5GlcNAc2 intermediate in the Golgi (Stanley,
essential for cell survival when glutamate is absent 1981). This enables the production of correctly