CFPS Disulfide Premium - Beta release

CFPS Disulfide Premium - Beta release

$575.00 USD
Volume

Ginkgo’s CFPS Disulfide Premium kit is tailored for the production of proteins containing disulfide bonds. It enables the synthesis of functional disulfide bonded targets that can’t properly express with standard CFPS, including antibody fragments, multi-domain therapeutic enzymes, and more.

The kit is an E. coli transcription/translation system optimized to produce proteins from a T7 RNA Polymerase promoter under redox conditions that facilitate disulfide bond (DSB) formation. Reactions consist of a bacterial cell lysate, a reagent mix containing all components required for protein production, and a DSB Booster that drives formation and correct pairing of DSBs in the target protein. The user must simply provide a DNA template that encodes the protein of interest under control of a T7 promoter. Reactions are assembled on ice, incubated at 30°C for 4 - 18 hours, and can be analyzed in solution or after purification.

The system has been optimized for proteins carrying 1 to 9 disulfide bonds and validated across protein classes spanning that range:

  • Antibody fragments: VHHs and scFvs (1–2 disulfide bonds), expressed with >95% DSB confirmed via intact mass spectrometry)
  • Reporter enzymes: Gaussia luciferase (5 disulfide bonds), supplied as the kit's positive control
  • Multi-domain therapeutic enzymes: reteplase (9 disulfide bonds), a plasminogen activator that is notoriously difficult to express solubly in conventional E. coli systems

Contents:

  • DSB E. coli Lysate
  • DSB Reagent Mix (3.3x)
  • DSB Booster (20x)
  • Control Gaussia Luciferase (GLuc) Plasmid (100 nM)

Technical Information:

  • Optimized for targets containing 1 to 9 disulfide bonds.
  • Compatible with both plasmid DNA and linear DNA templates. Eliminate time-consuming cloning steps by using linear DNA directly.
  • Expression is driven by a T7 RNA polymerase promoter.
  • Reaction volumes can be scaled up or down proportionally from the standard recommended 20 uL setup.
  • Includes a Gaussia luciferase (Gluc) positive control to verify successful CFPS.
  • Incubation at 30 °C for 4 to 18 hours is ideal across various protein classes (although target-specific optimization may improve titers).
  • Kit is stored at −80 °C. GLuc Control Plasmid at −20 °C.
  • The kit ships on dry ice.
  • This beta release is not compatible with downstream purification via His-tag (IMAC/Ni-NTA) affinity. Use an alternative affinity tag such as FLAG or Strep-II, or recover protein by size-exclusion or ion-exchange chromatography.
  • This kit is for research use only and is not for use in diagnostic or therapeutic procedures.

Quick Start Guide

CFPS Disulfide User Manual

Terms of Use

Safety Data Sheet

Cell-Free Protein Synthesis kits are for research use only. Administration to human and other animals and use in the clinic, for diagnostic purposes, and other uses are prohibited. Do not use in foods or for domestic purposes.

Do you have questions about our kit or want a consult on how to best use Ginkgo's CFPS kits? Are you interested in a custom order? Reach out to us at [email protected]
CFPS Disulfide Premium - Beta release
  • CFPS Disulfide Premium - Beta release
  • CFPS Disulfide Premium - Beta release

CFPS Disulfide Premium produces VHH domains above 100 mg/L across a five-member panel. Five VHH sequences directed against ADRA1A, IL-7Rα, insulin receptor, PD-L1, and SARS-CoV-2 spike RBD were expressed as Strep-tag®II fusions in CFPS Disulfide Premium reactions at 30 °C for 18 h. Titer (mg/L, bars) was determined by A280 following affinity purification. Disulfide bond formation (%DSB, circles) was quantified by mass spectrometry, comparing intact and reduced samples. Antigen binding was assessed on crude reaction products by biolayer interferometry (Gator Bio).

VHH expression, DSB formation and binding

  • Achieves robust cell-free expression yields >100 mg/L across diverse clinical benchmark VHH sequences.
  • Achieves close to 100% correct disulfide bond formation across targets with complex structure.
  • Confirmed positive binding responses against multiple antigens directly from CFPS lysate (no purification necessary).

CFPS Disulfide Premium supports scFv expression across a diverse six-member panel. A panel of six clinically-derived scFv sequences targeting PD-L1 (avelumab, durvalumab), SARS-CoV-2 spike RBD (FD20, PR1077), and IL-7Rα (2B8, 4A10) were expressed in CFPS Disulfide Premium Kit reactions at 25 °C for 8 h. Titers were measured post affinity purification.

More antibody fragments - scFv expression

  • Titers range from 25 to 103 mg/L on identical reaction chemistry, so the scFv identity is the main titer driver.
  • Heavy (VH) and light (VL) variable domains fused with a linker, each containing an intra-domain disulfide bond. No strain engineering required. 

Functional Gaussia luciferase expression is dependent on the disulfide-enabling environment of the CFPS Disulfide Premium system. GLuc was expressed with either the CFPS Disulfide Premium Kit or standard CFPS, and the crude reactions were assayed directly. Left, GLuc luminescence: CFPS Disulfide Premium reached approximately 1.1 × 10⁶ RLU, while standard CFPS remained at assay background. Right, GLuc titer back-calculated against a purified GLuc standard curve, yielding ~20 mg/L. Error bars are the standard deviation (n = 3).

High-level expression of go-to DSB reporter protein

  • Ginkgo CFPS Disulfide Premium yields ~ 20 mg/L of Gaussia luciferase.
  • No activity is observed using traditional CFPS.  
  • Use luciferase reaction as a positive control (plasmid included in the kit).

Active reteplase requires the disulfide-forming CFPS Disulfide Premium system. Reteplase was expressed for 18 h at 30 °C with Ginkgo’s CFPS Disulfide Premium Kit or standard CFPS, and the crude reaction products were assayed directly with a fluorogenic AMC tPA substrate. Left, AMC release kinetics over 60 min; traces are the mean of n = 3 reactions and shaded bands show ± 1 SD. Right, Reteplase activity back-calculated from the linear portion of each trace against a tPA standard curve. CFPS Disulfide Premium delivered approximately 7,000 U/mL, whereas standard CFPS gave no activity above assay background. 

Functional expression of reteplase - 9 disufide bonds

  • Ginkgo CFPS Disulfide Premium yields ~7,000 U/mL of reteplase.
  • No activity is observed using traditional CFPS.  
  • The readout is activity, not protein mass, so values indicate correctly folded enzyme.
  • Assay directly from the crude reaction. No need for purification or refolding.