Biomedical Engineering Reference
In-Depth Information
Figure
3.3. Time course of P. pastoris expression and secretion of recombinant EBA-175 RII by
shake flask fermentation. Supernatants were characterized by SDS-PAGE 4-20% gradient gel
analysis using Coomassie blue stain under nonreducing conditions. Doublet primarily represents
expression of N-linked and non-N-linked EBA-175 RII proteins. Control protein was purified
baculovirus-expressed EBA-175 RII [31].
72.7% to 43.3% and the potential internal polyadenylation sites were removed. The
synthetic gene encoding EBA-175 RII was cloned into the pPICZ
A vector and
transformed in P. pastoris host strain X33 following standard procedures [33]. The
pPICZ
a
-factor secretion signal from S. cerevisiae for directing
secreted expression of the recombinant protein. Transformants were screened by shake
flask fermentation using methanol as the sole carbon source for induction of the alcohol
oxidase I gene promoter. The results of shake flask fermentation for one clone are shown
in Fig. 3.3. P. pastoris expressed EBA-175 RII as a secreted product, which was observed
as a doublet; the lower band migrated at the expected molecular mass as determined by
Coomassie blue-stained SDS-PAGE gel analysis. For preclinical studies, recombinant
EBA-175 RII protein was purified by a series of column chromatography steps including
cation exchange, mixed mode hydrophobic interaction, and a second cation exchange
column [33]. An example of the purified recombinant EBA-175 RII protein is shown in
Fig. 3.4. The identity of the recombinant protein was determined by other biochemical
and biophysical techniques, including erythrocyte binding assay (Fig. 3.6), and immu-
noblot with conformation-dependent monoclonal antibodies [34].
EBA-175 RII protein contains five putative N-linked glycosylation sites based on
the conservation of the amino acid sequence NxS/T. The locations of the putative sites
are shown in Figs 3.1 and 3.5. Analysis of the purified recombinant EBA-175 RII
protein by carbohydrate analysis demonstrated the presence of N-linked carbohydrates,
that is, 20% or one in five sites randomly appeared to contain N-linked glycosylation
with MAN
14-16
GlcNAc
2
[33]. The native EBA-175 RII domain does not contain
N-linked glycosylated moieties due to the inability of P. falciparum parasites to
N-glycosylate proteins [35]. The presence of the N-linked carbohydrates could alter
the immunogenicity of the recombinant protein. Such an observation had been made for
another P. falciparum blood-stage protein identified as the 42 kDa fragment of the
a
A vector includes the
a
