Last data update: 24 November 2020 04:18 CET
Plasmid name: pPICZTS (LMBP 4420)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Trypanosoma cruzi trans-sialidase gene (TS); mature sequence
Myc epitope; C-terminal
Histidine tag (His-tag); C-terminal
|Promoter:||Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Saccharomyces cerevisiae translation elongation factor 1α promoter (TEF1α)
Synthetic prokaryotic EM7 promoter
|Terminator:||Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1)
|Selection marker:||Bleomycin (bleo; zeomycin (zeo; Zeocin); phleomycin (phleo))|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli; preferably recombination-deficient and endonuclease A deficient strains
Pichia pastoris; integrative
|Parental clone:||pPICZB; pPIC9-TSE|
|Further information:||This plasmid was constructed as follows: 1) The intermediate plasmid pPICZfragTS was constructed by inserting a KpnI-XbaI PCR fragment of the pPIC9-TSE vector, containing the 3' fragment of the mature T. cruzi TS gene, into the KpnI-XbaI opened pPICZB vector. 2) The PmeI-KpnI fragment of pPIC9-TSE, containing the ppMF signal sequence of the S. cerevisiae MFα1 gene fused to the 5' fragment of the mature T. cruzi TS gene, was then inserted into the PmeI-KpnI opened pPICZfragTS vector.
This plasmid is useful in P. pastoris strains for secreted expression of trans-sialidase under control of the strong, highly-inducible P. pastoris AOX1 promoter.
At the fusion junction between the ppMF sequence of the S. cerevisiae MFα1 gene and the mature TS gene, a (Glu-Ala)-dipeptide and an additional (but not essential) Tyr-residue are present. During secretion of the protein, the (Glu-Ala)-dipeptide is removed by a dipeptidase.
The zeocin resistance gene is driven by the synthetic prokaryotic EM7 promoter in E. coli and by the S. cerevisiae TEF1α promoter for selection in P. pastoris.
The presence of two P. pastoris AOX1 regions (the AOX1 promoter and the AOX1 terminator) allows integration of the linearized vector into the Pichia genome via homologous recombination at the AOX1 locus.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727204.1.
|EMBL Accession number:||AJ276679, view at EMBL, GenBank, DDBJ
LT727204.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||17/03/2008|
Primers used for PCR on pPIC9-TSE: forward: 5' GAG.ACG.GGT.AAG.AGG.TAC.CAC.GTG 3' KpnI reverse: 5' ACG.TTC.TAG.ACC.CAA.TTC.CTT.CGT.GTC.GCT.GCT.GCT.GTC 3' XbaI Nucleotide sequence around the start of the trans-sialidase gene: -- ppMF ---> | || || -- mature TS --> 5' ... GGG.GTA.TCT.CTC.GAG.AAA.AGA|GAG.GCT||GAA.GCT||TAC.GTG.CTG.GCA.CCC ... 3' Gly Val Ser Leu Glu Lys Arg|Glu Ala||Glu Ala||Tyr Val Leu Ala Pro XhoI | || ||------- HindIII SnaBI/PmlI fusion | : Kex2 cleavage site. ||: Dipeptidase cleavage. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Alw44I/KpnI, BglII and XbaI.|
|History of deposit:||This plasmid was deposited by S. Ryckaert(1) (2) and Prof. Dr R. Contreras(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Ryckaert et al., J. Biotechnol. 119 (2005), 379-388 [PMID: 15982773] [DOI: 10.1016/j.jbiotec.2005.04.010]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061|
|Host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Related host reference:||Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
|Cultivation medium:||LB-Lennox + zeocin (25 μg/ml)|
|Other culture collection numbers:||-|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.