Last data update: 24 November 2020 04:18 CET
Plasmid name: pPIC9-TS (LMBP 4173)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Trypanosoma cruzi trans-sialidase gene (TS); mature sequence
|Promoter:||Pichia pastoris alcohol oxidase 1 promoter (AOX1)|
|Terminator:||Pichia pastoris alcohol oxidase 1 terminator (AOX1)|
|Selection marker:||Ampicillin (amp)
Pichia pastoris HIS4; auxotrophic
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli
Pichia pastoris; his4(-), integrative
|Parental clone:||pCAGGS-TS; pMa58; pPIC9|
|Further information:||The plasmid was constructed as follows: 1) The Trypanosoma cruzi trans-sialidase coding sequence (TS) was isolated as a StuI/MscI fragment from pCAGGS-TS and ligated into the SmaI opened pMa58 vector, resulting in the intermediate construct pMa58TSvoll; 2) The start codon of the mature TS gene was extinguished by site-specific mutagenesis using the synthetic linker 5'-CCGGGTGCCAGCACGTGGGGGAATTCCC-3'; 3) Finally, the mature TS gene was isolated as a PmlI (partial)/XbaI fragment and ligated into the SnaBI/AvrII opened pPIC9 vector, leading to pPIC9-TS.
This plasmid is useful in P. pastoris strains for secreted expression of trans-sialidase under control of the strong, highly-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter.
At the fusion junction between the prepro secretion signal sequence (ppMF) of the Saccharomyces cerevisiae α-mating factor 1 gene (MFα1) 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 presence of the P. pastoris HIS4 gene and two P. pastoris AOX1 regions (the AOX1 promoter and the AOX1 3' flanking region) allows integration of the linearized vector into the Pichia genome via homologous recombination at the his4 locus or AOX1 locus respectively.
Other name of the plasmid is pPIC9-TSj2.
|EMBL Accession number:||-|
|Latest sequence update:||25/08/2000|
Nucleotide sequence around the start of the trans-sialidase gene: -->trans-sialidase ------ ppMF -------> | || || -------- Gly Val Ser Leu Glu Lys Arg|Glu Ala||Glu Ala||Tyr Val Leu Ala Pro 5' GGG.GTA.TCT.CTC.GAG.AAA.AGA|GAG.GCT||GAA.GCT||TAC.GTG.CTG.GCA.CCC. 3' XhoI | || ||------- HindIII SnaBI/PmlI fusion |: Kex2 cleavage site. ||: Dipeptidase cleavage. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: AvaI, BglII and XhoI.|
|History of deposit:||This plasmid was deposited by Prof. Dr N. Callewaert(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Laroy et al., Protein Expr. Purif. 20 (2000), 389-393 [PMID: 11087678]
|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 + ampicillin (100 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.