Biomedical Engineering Reference
In-Depth Information
merozoite surface protein 1 (MSP1
42
) [36]. Recombinant forms of MSP1
42
protein were
expressed in transgenic mice either with or without a putativeN-linked glycosylation site
present near a critical enzymatic cleavage site [37]. These two recombinant forms of
MSP1
42
were purified and tested for their capacity to induce immunological protection
against a blood-stage challenge infection using a primate malaria model. Despite both
forms having a similar immunogenicity profile, the recombinant N-linked glycosylated
MSP1
42
protein failed to induce any protective response compared to the recombinant
non-N-linked glycosylated protein [36].
The observation that the recombinant N-linked glycosylated MSP1
42
protein failed
to protect Aotus monkeys against a challenge infection impacted the malaria vaccine
community in such a manner that it became a necessity to remove putative N-linked
glycosylation sites from recombinantly expressed proteins derived from eukaryotic
expression systems such as P. pastoris. This impacted the QbD approach for the devel-
opment of EBA-175 RII in one important manner. It provided for the improvement of a
CQA by eliminating the presence of N-linked carbohydrates, which subsequently lead to
an increase in the quantity of the bulk drug substance. However, it also added a level of
uncertainty to the integrity of the recombinant protein's structure, which could alter its
immunogenicity. In the case of EBA-175 RII, it was decided to mutate the putative
N-linked glycosylation sites and characterize the recombinantly expressed protein for its
functional activity, that is, binding erythrocytes (given subsequently) and induction of
inhibitory antibodies.
P. pastoris Expression and Characterization of Non-N-Glycosylated
EBA-175 RII (RII-NG).
EBA-175 RII was expressed and purified as a non-N-
glycosylated protein (identified as RII-NG) using the same expression plasmid, shake
flask fermentation, and purification strategy, already described. Themammalian-encoded
synthetic gene was used as a template. The five putative N-linked glycosylation sites
identified within EBA-175 RII were mutated to remove the consensus sequence NxS/T.
The following hierarchy was used to introduce the point mutations. First, homologous
deduced amino acid sequence alignments from other P. falciparum strains expressing
EBA-175 were evaluated for naturally occurring point mutations. Second, paralogous
[38-40] and orthologous [41] deduced amino acid sequence alignments were evaluated
for possible amino acid substitutions. Finally, a “best guess”was used such that asparagine
is mutated to glutamine and serine/threonine is mutated to alanine. The amino acid
sequence positions are shown in Fig. 3.1. Even though the strategy described above was
used, none of the alignments provided any help in the selection of the “best” amino acid for
substitution. Thus, the gene was mutated such that either the first amino acid (N-Q) or
third amino acid (S/T-A) was substituted to remove the consensus sequence NxS/T.
Transformants were screened by shake flask fermentation and analyzed by Coomassie
blue-stained SDS-PAGE gel. A production clone was selected and used to produce
purified recombinant EBA-175 RII-NG protein. A Coomassie blue-stained
SDS-PAGE gel showing a comparison of EBA-175 RII and RII-NG is shown in
Fig. 3.4. Except for the absence of N-linked glycosylation, the biophysical characteri-
zation of purified EBA-175 RII-NG demonstrated that it appeared similar to EBA-175
RII protein, as determined by immunoblot with conformation-dependent mAbs [34] and
