Biomedical Engineering Reference
In-Depth Information
victoria, which fluoresces spontaneously without added substrate. There
are a number of variants of GFP that have been genetically engineered
to fluoresce at different wavelengths, including yellow fluorescent protein
(YFP) and cyan fluorescent protein (CFP).
Expression vectors
Plasmids can be used to express recombinant proteins from both
prokaryotic and eukaryotic cells. These “expression vectors” are de-
signed to allow the expression of recombinant DNA sequences in an
appropriate host cell. Expression cloning can be used to express a pro-
tein at high levels, analyze its function, and identify other proteins with
which it interacts, or to investigate its subcellular localization. Expres-
sion vectors are similar to conventional plasmids, except the MCS is
downstream of an inducible promoter.
Expression vectors are often designed so that the expressed gene
is cloned in-frame with a “tag”, at either the or end. The tag is a
sequence that is added to the protein of interest that facilitates both
purification and detection. The tag can be detected with an antibody
or other specific reagent which then facilitates purification of the fusion
protein (a protein that contains two different types of sequences) using
affinity chromatography. Some vectors are constructed with an enzyme
cleavage site between the expressed protein and the tag, enabling re-
moval of the tag after purification of the fusion protein. Common tags
include the FLAG tag, which is an 8 amino acid sequence for which spe-
cific antibodies are available; the His tag, which is a sequence encoding
6 consecutive histidine residues that binds nickel with high affinity; and
the GST (glutathione-S-transferase) tag, which binds glutathione with
high specificity. Each of these types of fusion proteins can be isolated
using affinity columns containing the corresponding antibody or ligand,
making expression cloning and purification possible even without having
an antibody for the protein being cloned.
Eukaryotic expression vectors
These are plasmids that have been adapted for the expression of
recombinant proteins in eukaryotic cells. These vectors require the in-
clusion of a promoter that is activated in the cell in which the gene is to
be expressed. Frequently, a promoter that is responsive to ubiquitously
expressed transcription factors is included in these vectors, so that they
can be used for expression studies in virtually all eukaryotic cells. Com-
mon promoters include the beta actin promoter and the cytomegalovirus
(CMV) promoter. In addition to a promoter, these vectors may contain
Search WWH ::
Custom Search