Synthetic Gene DataBase
 

Synthetic Gene 210


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID194210
GenBank AccessionU65407AY599500
GenBank GI157553147078048
Gene NameAMA1-3D7EcAMA1-3D7
Gene Length (bp)18691611
SpeciesPlasmodium falciparum (malaria)Escherichia coli
Strains3D7BL21 (DE3)
CDSatgagaaaattatactgcgtattattattgagcgcctttgagtttacatatatgataaac
tttggaagaggacagaattattgggaacatccatatcaaaatagtgatgtgtatcgtcca
atcaacgaacatagggaacatccaaaagaatacgaatatccattacaccaggaacataca
taccaacaagaagattcaggagaagacgaaaatacattacaacacgcatatccaatagac
cacgaaggtgccgaacccgcaccacaagaacaaaatttattttcaagcattgaaatagta
gaaagaagtaattatatgggtaatccatggacggaatatatggcaaaatatgatattgaa
gaagttcatggttcaggtataagagtagatttaggagaagatgctgaagtagctggaact
caatatagacttccatcagggaaatgtccagtatttggtaaaggtataattattgagaat
tcaaatactacttttttaacaccggtagctacgggaaatcaatatttaaaagatggaggt
tttgcttttcctccaacagaacctcttatgtcaccaatgacattagatgaaatgagacat
ttttataaagataataaatatgtaaaaaatttagatgaattgactttatgttcaagacat
gcaggaaatatgattccagataatgataaaaattcaaattataaatatccagctgtttat
gatgacaaagataaaaagtgtcatatattatatattgcagctcaagaaaataatggtcct
agatattgtaataaagacgaaagtaaaagaaacagcatgttttgttttagaccagcaaaa
gatatatcatttcaaaactatacatatttaagtaagaatgtagttgataactgggaaaaa
gtttgccctagaaagaatttacagaatgcaaaattcggattatgggtcgatggaaattgt
gaagatataccacatgtaaatgaatttccagcaattgatctttttgaatgtaataaatta
gtttttgaattgagtgcttcggatcaacctaaacaatatgaacaacatttaacagattat
gaaaaaattaaagaaggtttcaaaaataagaacgctagtatgatcaaaagtgcttttctt
cccactggtgcttttaaagcagatagatataaaagtcatggtaagggttataattgggga
aattataacacagaaacacaaaaatgtgaaatttttaatgtcaaaccaacatgtttaatt
aacaattcatcatacattgctactactgctttgtcccatcccatcgaagttgaaaacaat
tttccatgttcattatataaagatgaaataatgaaagaaatcgaaagagaatcaaaacga
attaaattaaatgataatgatgatgaagggaataaaaaaattatagctccaagaattttt
atttcagatgataaagacagtttaaaatgcccatgtgaccctgaaatggtaagtaatagt
acatgtcgtttctttgtatgtaaatgtgtagaaagaagggcagaagtaacatcaaataat
gaagttgtagttaaagaagaatataaagatgaatatgcagatattcctgaacataaacca
acttatgataaaatgaaaattataattgcatcatcagctgctgtcgctgtattagcaact
attttaatggtttatctttataaaagaaaaggaaatgctgaaaaatatgataaaatggat
gaaccacaagattatgggaaatcaaattcaagaaatgatgaaatgttagatcctgaggca
tctttttggggggaagaaaaaagagcatcacatacaacaccagttctgatggaaaaacca
tactattaa
atgattaattttgggagagggcagaactactgggaacatccataccaaaactctgatgtg
tacaggccaataaatgaacatcgtgaacatccaaaggaatatgaatacccattgcaccag
gaacatacttatcaacaagaagattccggggaagacgaaaacactttgcaacacgcatac
ccaattgaccacgaaggagctgaacccgcaccacaagaacaaaacttgttttccagcatc
gaaattgtcgaaagatctaactacatgggaaacccatggacggaatacatggcaaagtac
gatatcgaagaagttcatggttccggaattagagtcgatttgggggaagatgcggaagtc
gcggggacacaatacagacttccatccggtaagtgtccagtctttggaaagggaattatc
atcgagaactccaagacaacatttttgactccggtcgcgacggggaaccaatacttgaag
gatgggggatttgcgtttcctccaactgaacctctgatgtccccaatgactttggatgaa
atgagacatttttacaaggataacaagtacgtcaagaacttggatgaactgacattgtgt
tccagacatgcagggaacatgatcccagataacgataagaactccaactacaagtaccca
gcggtttacgatgacaaggataagaaatgtcatattttgtacatcgcagcgcaagaaaac
aacggacctagatactgtaacaaggacgaatctaagagaaatagcatgttttgttttaga
ccagcaaaggatatttcctttcaaaatttggtctacttgtctaaaaacgtcgttgataat
tgggaaaaggtttgccctagaaagaacttgcagaacgcaaagttcgggttgtgggtagat
gggaactgtgaagatattccacatgtcaacgaatttccagcaatcgatctttttgaatgt
aacaagttggtttttgaactgtctgcgtcggatcaacctaagcaatacgaacaacatttg
actgattacgaaaagatcaaggaaggattcaagaacaaaaatagagaaatgataaagtct
gcgtttcttcccacaggagcgtttaaggcagatagatacaagtctcatggaaaaggatac
aactgggggaactacaatactgaaactcaaaagtgtgaaatctttaacgtaaagccaact
tgtttgatcaatgacaagaactatatcgcgacaacagcgctgagtcatcccatagaagtt
gaaaataactttccatgttccttgtacaaggatgaaattatgaaggaaatagaaagagaa
tccaagcgaatcaagttgaacgataacgatgatgaaggtaacaagaagatcattgcgcca
agaatctttatctccgatgataaggactctttgaagtgcccatgtgaccctgaaatggtc
tctaactctacttgtaggttctttgtctgtaagtgtgtcgaaagacgtgcagaagtcact
tccaacaacgaagttgtcgttaaggaagaatacaaggatgaatacgcagatatccctgaa
cataagccaacatacgataagatgaagctcgagcaccaccaccaccaccac
5' End
3' End
NotesIt is a partial CDS (M18 to K546) and contains an additional LEHHHHHH sequence at the C terminus to facilitate nickel affinity purification.
Expression VectorNApET24d+
Assay MethodsNASDS-PAGE, Western blot, Coomassie blue staining
ResultsNot determinedSuccessful expression
Protein FunctionBlood cell invasion (antigen)
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodThe coding sequence of AMA1 gene (M18 to K546) was modified (N-linked glycosylation sites and codon
bias for GC-rich sequence) and optimized for expression in E. coli by normalizing their AT content
according to published values for E. coli codon bias.
Publication Author(s)Giersing B, Miura K, Shimp R, Wang J, Zhou H, Orcutt A, Stowers A, Saul A, Miller LH, Long C, Singh S.
Corresponding AuthorSanjay Singh
Corresponding AddressMalaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, Twinbrook I, Room 1210A, 5640 Fisher Lane, Rockville, Maryland 20852, USA.
Publication Year2005
Publication TitlePosttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to a malaria vaccine candidate.
AbstractRecombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.
JournalInfect Immun.. 73(7): 3963-70.
SummaryAMA1 genes from two P. falciparum strains were codon optimized for expression in E. coli and P. pastoris due to high AT content of the malaria genome. All four synthetic genes appeared to expressed at a detectable level.
Comments
Discussion http://www.evolvingcode.net/forum/viewtopic.php?t=503
PubMed ID15972483
Submitter NameWu, Gang
Submitter AddressDepartment of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 USA
Entry ConfirmationNo
 
 

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