Last data update: 24 November 2020 04:18 CET
Plasmid name: pPLT10HIs (LMBP 2589)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Escherichia coli outer membrane protein II gene (ompA); signal sequence
Human immunoglobulin γ3; heavy chain hinge region
|Promoter:||Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
|Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)|
|Terminator:||Phage T7 gene 10 terminator (T7g10)|
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli; use strains with a cI function, cIts for PL controlled expression|
|Parental clone:||pPLT10HB2Mms; pMacBLAL2HI|
|Further information:||The plasmid was constructed by substituting the SacI (nucleotide position 167) - BstXI (blunted with T4 DNA polymerase) fragment of pPLT10HB2Mms, encoding the mutated mature human β2 microglobulin gene, by the DraI (nucleotide position 1700) - SacI fragment of pMacBLAL2HI, encoding the human γ3 hinge region. In this way, the human γ3 hinge region (HI) was fused in phase to the signal sequence of the E. coli outer membrane protein II (sompA).
The translation of the fusion protein terminates two amino acids behind the fourth hinge region (H4), caused by the presence of a termination codon in the CH2 domain.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene, as well as a cI function: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857]. Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
The sequence of the inserted hIG3 fragment was taken from 'GENBANK Release 55; Locus: HUMIGHAF' and contains some non-identified nucleotides (indicated by N).
Other name of the plasmid is pPLT10HIsT.
|EMBL Accession number:||J00231, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||23/01/1997|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061(λ)|
|Host reference:||Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
|Related host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|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.