Last data update: 24 January 2024 16:39 CET
Plasmid name: pSE380rPDI (LMBP 3051)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3051.gb
(View with Genome Compiler) p3051.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Rat protein disulfide isomerase cDNA (rPDI) Escherichia coli lac repressor gene (lacI) |
| Promoter: | Escherichia coli hybrid tryptophan/lacUV5 promoter (trc) Escherichia coli lac repressor promoter; mutant (lacI(q)) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| Terminator: | Escherichia coli rrnB operon T1 terminator Escherichia coli rrnB operon T2 terminator |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pSE380; ppdi100 |
| Further information: | To construct pSE380rPDI, the rat protein disulfide isomerase (rPDI) gene of ppdi100 was cloned into the multiple cloning site downstream from the Escherichia coli hybrid tryptophan/lac operon (trc) promoter in pSE380: 1) ppdi100 was first digested with Nsp(7524)I (isoschizomer of NspHI), blunted with T4 DNA polymerase and further cleaved with BglII; 2) Then, the 1535 bp fragment containing the 'truncated' rPDI gene was ligated to the 4350 bp vector fragment of pSE380, previously digested with NcoI, filled in with Klenow DNA polymerase and then cut with BglII. The mature rPDI gene, preceded by its own signal sequence, is expressed under control of the strong, IPTG-inducible trc promoter, followed by the lacZ ribosome binding site. This expression vector is further characterized by the presence of the rrnB 5S gene and the strong rrnB transcription terminators T1 and T2 downstream from the rPDI gene to prevent read-through transcription. The plasmid also contains the E. coli lacI(q) cassette, leading to overproduction of the lac repressor (LacI), to repress the trc promoter in order to expand the host range of the vector to non-lacI(q) strains (e.g. MC1061). pSE380rPDI was used for the bacterial production of enzymatically active rat protein disulfide isomerase, which was released from the periplasm by cold osmotic shock. This production was always accompanied by the synthesis of a small peptide of about 21 kDa (rPDIf) due to internal translation initiation within the rat PDI gene. Replacement of the presumed internal start codon by CTC eliminated the aforementioned phenomenon completely. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727424.1. The nucleotide sequence of the rPDI cDNA corresponds with the EMBL Nucleotide Sequence Database (Release 36; Accession number X02918). |
| EMBL Accession number: | X02918, view at EMBL, GenBank, DDBJ LT727424.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 19/03/1992 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglI/BglII, EcoRI/PstI and NotI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. De Sutter(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | De Sutter et al., Gene 141 (1994), 163-170 [PMID: 8163184] |
| 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) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSE380rPDI (LMBP 3051) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter and was published in De Sutter et al., 1994. |
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.