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the GH signal sequence. These vectors were then transfected
into Chinese hamster ovary (CHO) cells and stable clones
generated. These clones were adapted to suspension culture
and grown in protein-free media; the expressed LR-fusion
was secreted into the growth media.
The LR-fusions were purified from the growth media by
affinity chromatography using columns containing immo-
bilized anti-GH antibodies.
15.4 ANALYSIS OF THE LR-FUSIONS
15.4.1 Characterization
The purified GH-LRv0 was screened by ELISA using 16
conformationally sensitive anti-GHBP monoclonal antibod-
ies; the affinities of these antibodies were similar those
obtained against serum-derived GHBP.
Coomassie staining of a SDS-PAGE gel showed the LR-
fusion protein separated as a doublet at a mass of
FIGURE 15.2 A schematic diagram of the expressed GH LR-
fusions; GH-LRv0 contains extraneous sequence that occurred as a
consequence of the cloning strategy used to synthesize the gene for
expression. The extraneous sequences at the N- and C-termini were
removed to produce GH-LRv1. The restriction sites used to insert
the linker into the molecule were removed to generate GH-LRv2.
GH-LRv3 was then synthesized by removing the linker region.
75 kDa
(Figure 15.3A). Immunoblotting using anti-GH antibody as
a probe confirmed this band-pattern. The formula mass of
the GH-LRv0 is
55 kDa; it was assumed that differential
glycosylation of the 55 kDa protein would account for the
two bands observed at 75 kDa.
Native-PAGE gels of the LR-fusion also showed two bands
(Figure 15.3B); however, it was thought that the differences in
sizes between the two bands were unlikely to be simply due
differential glycosylation. The “fast” and “slow” bands from
the native gel were excised and reanalyzed by SDS-PAGE
under reducing conditions; both the reanalyzed “fast” and
“slow” bands showed similar band-patterning to that of the
original SDS-PAGE, that is, a doublet of bands at 75 kDa
separated by
structure [22] and so we questioned whether there was a need
for the (Gly 4 Ser) 4 linker. We, therefore, linked the C-termi-
nus of GH directly to the N-terminus of GHRec, to generate
variant 3 (GH-LRv3).
15.3 EXPRESSION AND PURIFICATION
The genes encoding the LR-fusions were cloned into an
expression vector under the control of a CMV promoter and
5 kDa (Figure 15.3C); this result suggested that
FIGURE 15.3 (A) SDS-PAGE of GH-LRv0, followed by coomassie staining (CS) or Western
blotting (WB) using an anti-GH antibody. GH-LRv0 is 75 kDa and resolves into two bands, 5 kDa
apart. (B) Native gel of GH-LRv0 showing that there are two protein forms; fast (F) and slow (S). (C)
The individual F and S bands were excised from the native gel and subjected to SDS-PAGE under
reducing conditions, followed by Western blotting using an anti-GH antibody. When rerun on a SDS-
PAGE gel the two bands, F and S, run at 75 kDa and separate as a doublet, suggesting that they both
comprise the 75 kDa LR-fusion, which may exist in an equilibrium of monomers and dimers under
native conditions. Source: This research was originally published in Nature Medicine, Reference
[13].
Nature Publication Group.
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