Biomedical Engineering Reference
In-Depth Information
TABL E 3.3. Antibodies Against Recombinant RII and RII-NG Proteins Block Function
of EBA-175
% Growth Inhibition
of Erythrocyte Invasion
b
RII ELISA Titer
a
Recombinant
P. pastoris
RII Vaccine
c
RII
Capture
Antigen
RII-NG
Capture
Antigen
FVO
Parasites
3D7
Parasites
Rabbit No.
74
RII
937,296
623,374
22
25
75
1,656,000
1,442,000
29
ND
76
RII-NG
355,233
562,888
20
23
77
244,534
407,154
18
17
a
Rabbits 74/75, 76/77, and controls were immunized thrice with 100
m
g recombinant protein in Freund's
adjuvant or adjuvant alone for controls.
b
Interpolated reciprocal RII ELISA titers are reported for anODof 0.5 using either RII or RII-NG for the capture
antigen.
c
%Growth inhibition reported using purified rabbit IgG at 1.0mg/mL compared to control. All values were
statistically significant p
<
0.05.
3.3.2 P. pastoris Expression of Apical Membrane Antigen 1 (AMA1):
Codon Selection
In the following example, the selection of the codon usage is discussed for another
important malarial vaccine target, that is, recombinant AMA1 3D7 protein. Amolecular
schematic of the protein is shown in Fig. 3.7. Two different approaches for the molecular
design of the synthetic AMA1 3D7 gene were used. The Malaria Group from EntreMed,
Inc. selected to use a mammalian codon optimized gene while MVDB selected to use a
P. pastoris codon optimized gene for expression in P. pastoris. A codon usage table
similar to that used by the two groups is shown in Table 3.1, which compares the optimum
codons used by P. falciparum to that of H. sapiens (mammalian) or P. pastoris.A
comparison of the native, mammalian codon optimized and P. pastoris codon optimized
genes is shown in Fig. 3.8. A total of 525 out of 1560 nucleotides (33.7%) were altered in
the synthetic mammalian gene; a total of 306 out of 1575 nucleotides (19.4%) were
altered in the P. pastoris-encoded gene. The A
T content of the AMA1 genes changed
from 69.4% for the native gene to 38.0% for the mammalian and 58.8% for P. pastoris
genes. Most of the nucleotide changes occurred in the third base position. Most striking is
that the long stretches of poly A's were removed to prevent the formation of truncated
mRNA transcripts. Six putative N-linked glycosylation sites were identified and their
amino acid sequence and position are shown in Fig. 3.7. In both of these synthetic genes,
the codons were changed to remove the putative N-linked glycosylation sites using a
similar methodology to that discussed above for EBA-175 RII. Appropriate restriction
enzyme sites were modified to allow cloning in either pPICZ
รพ
A (Narum, Merchant,
Fuhrmann, and Kumar, unpublished) or pPIC9K [17]. Expression plasmids were
a
