Biomedical Engineering Reference
In-Depth Information
Figure
3.7.
Scheme of gene structure of AMA1 showing division of domains I-III and other
domains (upper panel). The lower panel shows the native AMA1 3D7 amino acid sequences in
which the putative N-linked glycosylation sites are identified (NxS/T) by amino acid position
using the sequence identified by accession number: U65407. The six amino acid substitutions
used for expression of the synthetic P. pastoris or mammalian codon optimized gene are shown
in red. The solid black bar represents the region recombinantly expressed in
. The
position of putative N-linked glycosylation sites are noted by asterisks. CD, cytoplasmic domain;
SS, signal sequence; TM, transmembrane domain. (See the insert for color representation of this
figure.)
P. pastoris
linearized and appropriate strains (X33 or GS115) of P. pastoris were transformed and
placed under selection pressure. Recombinant AMA1 protein production clones
were identified using either the synthetic mammalian codon optimized gene (Narum,
Merchant, Fuhrmann, and Chen, unpublished) or P. pastoris codon optimized gene [17].
Recombinant AMA1 3D7 protein was produced by both groups. Recombinant
AMA1 3D7 protein produced by MDVB [17] was extensively characterized using most
of the analytical methods shown in Table 3.2. For the purpose of this discussion, there are
two important CQAs that were observed in both the recombinant AMA1 3D7 proteins,
evenwith themutations to remove the six putativeN-linked glycosylation sites (Fig. 3.7).
First, both recombinant AMA1 3D7 proteins induced growth inhibitory antibodies as
detected by inhibition of parasite growth in vitro (Narum and Haynes, unpublished and
Ref. [17]). Second, both recombinant proteins maintained a conserved structural epitope
as determined by immunoblotting with a conformation-dependent monoclonal antibody
identified as 4G2 (data not shown).
To conclude, expression of numerous malarial proteins by P. pastoris has been made
possible by changing the codon usage of the native gene that prevents premature
termination of mRNA transcription. The significance of which optimized codons to
use, mammalian, P. pastoris, or other, must be empirically tested. In addition, there are
other factors, which are not discussed here, that may be important to consider such as the
expression vector or promoter being used, potential for formation of mRNA hairpins, as
