FabRICATOR (IdeS) is an IgG-specific cysteine protease that digests antibodies or Fc-fusion proteins at one single amino acid position below the hinge region generating Fc and F(ab’)2 fragments. FabRICATOR is lyophilized in various formats for digestion of 100 ug – 25 mg of antibodies or Fc-fusion proteins.
| Application | |
|---|---|
| Product Group | |
| Source/Origin | Cloned from S. pyogenes, Produced in E. coli (His-tagged), 37.7 kDa |
| Product Composition | |
| Digestion Site | |
| Specificity | |
| Substrates | Human IgG1, Human IgG2, Human IgG4, Monkey IgG, Rabbit IgG, Rat IgG, Sheep IgG |
| Reaction Conditions | 30 minute reaction at 37°C according to protocol specifications |
| Digestion Buffers | PBS (pH 6.0 – 8.0), TBS (pH 7.0 – 8.0), MES (pH 5.5 – 6.5), HEPES (pH 7.0 – 8.0), Ammonuim Bicarbonate (pH 6.0 – 7.0), Sodium Acetate (pH 6.0) |
| Buffer Compatibility | |
| Inhibitors | |
| Unit Definition |
Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.
We have no stability data for storage at -20°C or -70°C. However, we have customer reporting that they successfully stored reconstituted FabRICATOR at -20°C or -70°C for a couple of months. Genovis cannot guarantee the activity of the enzyme, but these storage conditions might be OK depending on the intended application.
Yes, FabRICATOR should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabRICATOR digests human IgG just below the hinge (…CPAPELLG / GPSVF…), leaving the hinge on the fusion-part.
It is possible to use FabRICATOR with low concentrations of IgG. Low concentrations of IgG will lead to decreased reaction rate, so for concentrations below 0.5mg/mL it is therefore recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of over-digestion and the incubation time can be prolonged to O/N if necessary.
Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.
No. According to the following reference, FabRICATOR does not digest IgM. Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).
In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.
If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 – 750 units of FabRICATOR.
FabRICATOR is a very specific enzyme and small changes in the sequence in or around the digestion site (…CPAPELLG / GPSVF…) may reduce or completely impair the ability to digest the IgG. It is very difficult to predict the outcome for each specific modification. It might work, but we cannot guarantee it unfortunately, the only way of knowing is to perform a digestion test. Some suggestions that are worth testing if you experience incomplete digestion: Increase the incubation time, increase the amount of added enzyme, use a PBS with greatly reduced salt concentration (5-10 mM instead of 140 mM) and pH 6.5 instead of 7.4.
We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.
Yes. FabRICATOR is compatible with EDTA. Make sure your pH is within 6-8.
FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: ..CPAPELLG / GPSVF..
Human IgG2: ..CPAPPVA / GPSVF..
Human IgG3: ..CPAPELLG / GPSVF..
Human IgG4: ..CPAPEFLG / GPSVF..
