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Plasmid for high yield production of recombinant murine IgG1 kappa mAbs
High-yield production of recombinant murine IgG1 kappa mAbs
pTRIOZ-mhIgG1e2 is designed for high-yield production of whole monoclonal antibodies (mAbs) with the use of a single plasmid. It consists of three cassettes encoding for the expression of the mAb heavy chain, light chain, and antibiotic selection with Zeocin™.
pTRIOZ-mhIgG1e2 expresses the constant regions of:
- The murine IgG1 heavy chain with the T252M mutation, increasing its affinity for Protein A and enabling efficient purification by affinity chromatography.
- The murine kappa light chain
Upstream of both of these regions are unique multiple cloning sites (MCS) that enable the insertion of the variable regions of any given mAb.
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Heavy chain cassette:
- Composite promoter: Aldehyde dehydrogenase (AldA) enhancer/ Human ferritin heavy chain gene (hFerH)
- Cloning sites: 5’- AgeI, MluI, EcoRV, BspHI, NheI, and Eco47III -3’
- Constant region: Murine IgG1e2
Light chain cassette:
- Composite promoter: Human cytomegalovirus immediate-early gene 1 ( hCMV ) enhancer / Human ferritin light chain gene (hFerL)
- Cloning sites: 5’- SgrAI, AscI, and PmeI -3’.
- Constant region: Murine kappa
Antibiotic resistance: Zeocin™ (selectable in both bacterial and mammalian cells)
- Plasmid construct has been confirmed using DNA sequencing and restriction analysis
- Purified by ion exchange chromatography
- 20 µg of lyophilized pTRIOZ-mIgG1e2 plasmid DNA
- 1 ml of Zeocin™ (100 mg/ml)
Product is shipped at room temperature
Upon receipt product should be stored at -20°C
Resuspended DNA should be stored at -20 ̊C and is stable up to 1 year.Back to the top
Cassette 1: mAb Heavy chain
- AldA enh/ hFerH: This composite promoter combines the human aldehyde dehydrogenase (aldA) enhancer and the core promoter of the human ferritin heavy chain gene.
- MCS1: To facilitate cloning of the variable heavy (VH) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes,
- mIgG1e2: The constant region of the murine immunoglobulin IgG1 heavy chain with the T252M mutation to increase protein A affinity for purification.
- βGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allow the efficient arrest of the transgene transcription.
Cassette 2: mAb Light chain
- hCMV enh / hFerL prom: This composite promoter combines the human cytomegalovirus (CMV) immediate-early gene 1 enhancer and the core promoter of the human ferritin light chain gene.
- MCS2: To facilitate cloning of the variable light (VL) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes,
- Murine κ light chain: The constant region of the murine kappa light chain
- SV40 pAn: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA
Cassette 3: Zeocin™ selection
- mCMV/hEF1-HTLV prom: This composite promoter combines mouse cytomegalovirus (mCMV) immediate-early gene 1 enhancer, the elongation Factor-1α (EF-1α) core promoter, as well as the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) type 1 long terminal repeat. The EF-1α promoter exhibits a strong activity and yields a long-lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance the stability of RNA.
- EM7 prom: This is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. EM7 is located within an intron and is spliced out in mammalian cells.
- Sh Ble gene: Resistance to Zeocin™ is conferred by the Sh ble gene from Streptoalloteichus hindustanus. The same gene confers resistance in both mammalian cells and E. coli.
- hEF-1alpha pAn: This provides a strong polyadenylation signal. InvivoGen uses a sequence that starts after the stop codon of the EF1 cDNA and finishes after a bent structure rich in GT.
General features: pTRIOZ
- 5’ UTR: The 5’ UTR enhances mRNA stability and protein translation.
- Ori: A minimal E. coli origin of replication.