Antibody

Customers may select the pTwist CMV BG WPRE Neo expression vector when they would like to use their own Fc region sequence (IgG3, Mouse IgG, or modified Fc) or have another type of Fc-fusion antibody to express.

Please remember to include a stop codon at the end of your coding sequence. If you have any design questions, please email igg@twistbioscience.com for help with your custom request

Twist supplies clonal gene glycerol stocks from the clonal genes synthesized for antibody production. During antibody production, the DNA is consumed for transfection; however, glycerol stocks are reserved and delivered along with the antibodies post-production. Please see out protocol for preparing fresh DNA from glycerol stocks. 

Please ensure you have the following sequence format for uploading variable regions:

• Removed signal leader peptide (if using pTwist vectors + Twist recommended signal leader peptide)
• Variable regions should not end with any CH1 starting sequence
• Sequences are divisible by three if your ORF is in line with your insertion site

You can use a Protein A or G solid phase to purify, which binds to the FC region of the antibodies, depending on species and isotype of IgG. Alternatively, you can use immobilized antigen (full length or short peptide antigen) to purify the antibodies. The method will depend on the downstream application and specificity of the antibody.

A leader or secretory signal sequence acts as a peptide signal bound by cellular machinery early in the translation process. This allows for the correct localization of the translation complex to the endoplasmic reticulum where the antibody chains can enter into the secretory pathway for protein assembly and secretion from the cell. The leader sequence is cleaved during the protein assembly process and is not a part of the final protein. Twist recommends the following leader sequence for both heavy and light chains when using our pTwist IgG expression vectors as these sequences have been tested and validated with our pTwist vectors in both Expi293™ and ExpiCHO™ cell lines:

VH: MRAWIFFLLCLAGRALA

VL: MRAWIFFLLCLAGRALA

The Kozak sequence directs the pre-initiation complex (PIC) and ribosome to the translation initiation site (start codon) and mediates ribosome assembly ensuring the correct protein sequence is translated.

The consensus Kozak sequence is generally considered as GCCGCCACCATGG, where ATG is the start codon (typically the start of the signal sequence). Twist recommends the version GCCGCCACC upstream of the start codon ATG.

You can find the pricing table for our custom antibody products in your account by clicking here.

If you need to contact your Account Manager regarding pricing, their contact information is available in eCommerce under Account.

Twist uses the industry standard cell lines, Thermo Fisher Scientific Expi293™ and Thermo Fisher Scientific ExpiCHO™

This cystein in a full IgG forms a disulfide bond with the LC constant region which is not present in the VHH-Fc fusion construct.

Research has shown that C-terminal lysine residues of the heavy chain (present on all IgG isotypes) are cleaved off once the IgG is in circulation. Studies have also shown that there is no functional impact of the lysine once it’s cleaved and it’s presence is actually inhibitory to allowing the antibody to reach full cytotoxicity potential: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622059/

Twist inspects every antibody product for yield (μg), purity (%), and size (kDa) under reducing conditions. Each antibody will be inspected via LabChip (CE-SDS) to inspect purity and size of the individual heavy and light chains. Yields are quantified using A280 readings. Supernatant samples are quantified using a titer determination method. In the finalized documentation, each purified antibody will have reported volume, yield, and concentration values in addition to percent purity and heavy chain and light chain sizing. Supernatant documentation will provide antibody titer expression levels of either low, medium or high expression levels.

We use commercially available PBS, please share catalog information with customer upon request:

• PBS 7.4 Cat# 21-031-CM Corning
• PBS 6.0 Cat# P1760 Teknova

• 96-deep well plates - custom plate maps available
• 1.4 mL micronic tube racks
• 2 mL tubes
• 4 mL tubes
• 10 mL tubes (for 8 mL supernatant only)

1 mL = ~220 uL volume, 15-300 µg

8 mL = ~750 uL volume, 15-3000 µg