pTRIOZ-mIgG1e2

pTRIOZ General plasmid map

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.

Specifications

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)

Quality control:

• Plasmid construct has been confirmed using DNA sequencing and restriction analysis
• Purified by ion exchange chromatography

Contents

• 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.

Details

PLASMID FEATURES

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.