pTRIOZ-hIgG4 (S228P)

pTRIOZ General plasmid map

High-yield production of recombinant human IgG4 kappa mAbs 

pTRIOZ-hIgG4 (S228P)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-hIgG4 (S228P) expresses the constant regions of:

• The human IgG4 heavy chain with the S228P mutation, ensuring the stabilization of the disulfide bonds in the core-hinge region of IgG4 molecules to reduce Fab-arm exchange. 
• The human 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, and NheI -3’
• Constant region: Human IgG4 (S228P)

Light chain cassette:

• Composite promoter: Human cytomegalovirus immediate-early gene 1 ( hCMV ) enhancer / Human ferritin light chain gene ( hFerL )
• Cloning sites: 5’- SgrAI, AscI, PmeI, NcoI, and BsiWI -3’.
• Constant region: Human 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-hIgG4 (S228P) 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, 
• hIgG4: The constant region of the human immunoglobulin IgG4 heavy chain with the core-hinge stabilizing S228P mutation.
• 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, 
• Human κ light chain: The constant region of the human 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.