Synthetic Gene DataBase
 

Synthetic Gene 168


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID155168
GenBank Accession
GenBank GI
Gene NameA-dmDT390-bisFv(G4S)DTop390-bisFv(G4S)
Gene Length (bp)00
SpeciesCorynebacterium diphtheriaePichia pastoris
StrainsGS115, X-33, KM71
CDS
5' End
3' End
NotesGene is a fusion of diphtheria toxin(DT), two sFv domains, and a (G4S)3 linker.
Expression VectorpnPICZ-alphapnPICZ-alpha
Assay MethodsSDS-PAGE, Western BlotSDS-PAGE, Western Blot
ResultsNo expressionHigh increase (10 mg/L, 10 fold more, signal sequences did not affect result)
Protein FunctionImmunotoxin which intoxicates T cells and halts protein synthesis.
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodThe natural gene was codon optimized for bias in P. pastoris. The overall AT content was removed,
with some contructs containing a preprp secretion sequence.
Publication Author(s)Woo, J. H.; Liu, Y. Y.; Mathias, A.; Stavrou, S.; Wang, Z.; Thompson, J.; Neville, D. M., Jr.
Corresponding AuthorDavid M. Neville Jr.
Corresponding AddressSection on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, 36 RM 1B08, 36 Convent Drive, Bethesda, MD 20892-4034, USA.
Publication Year2002
Publication TitleGene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris
AbstractThe bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin. However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae. This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P. pastoris under the control of AOX1 (alcohol oxidase 1) promoter. The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P. pastoris because of several AT-rich regions, which induce an early termination of transcription. After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture. No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes. Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation. The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography. The immunotoxin purified from P. pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line. Our results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.
JournalProtein Expr Purif. 25(2): 270-82.
SummaryPichia pastoris was transformed with the DTop390-bisFv(G4S) gene, a recoded version of the natural gene, A-dmDT390-bisFv(G4S), in order to increase the expression of protein in the fungus. The natural gene was codon optimized based on the codon usage of highly expressed P. pastoris genes. The natural gene did not yield any protein, most likely from a problem with transcription, but the recoded gene was able to produce a highly significant amount of protein. From this result, the recoding can be considered very successful since the recoded gene was able to produce protein whereas the natural couldn’t.
CommentsCDS of recoded gene and natural gene was not provided. The author(s) did not wish to have the recoded gene CDS made public.
Discussion http://www.evolvingcode.net/forum/viewtopic.php?t=535
PubMed ID12135560
Submitter NameQureshi, Imran
Submitter Address1000 Hilltop Circle Baltimore, MD 21250 USA
Entry ConfirmationNo
 
 

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