Is it possible to prepare Fab fragments from different antibodies using GingisKHAN Fab kit?

Yes. GingisKHAN is provided lyophilized. After reconstitution, the enzyme can be used for up to 4 different antibodies. The digestion is performed in solution and you can prepare Fab fragments from 4 x 0.5 mg human IgG1. The purification step is done with Capture Select columns and 4 columns are provided in the kit.

Can I digest Fc-fusion proteins using GingisKHAN (Kgp)?

Yes, GingisKHAN should be able to digest a Fc fusion protein if you have not fused the protein too close to the cleavage site and the Fc-part is from a human IgG1. GingisKHAN digests human IgG1 just above the hinge (…KSCDK / THTCPPCP…). If there are exposed Lysines in the fusion part, digestion might occur there as well.

Can I use GingisKHAN on other subclasses than human IgG1?

No. GingisKHAN is a protease that digests at exposed Lysines with no secondary structure restrictions. At the recommended reaction conditions, GingisKHAN digestion above the hinge has only been confirmed on human IgG1 with the exposed lysine above the hinge, (…KSCDK / THTCPPCP…). No digestion has been confirmed on other subclasses of human IgG except for several digestion sites in human IgG3.

What is the difference between FabALACTICA and GingisKHAN?

FabALACTICA (IgdE) and GingisKHAN (Kgp) are both cysteine proteases that digest IgG1 above the hinge to generate intact Fab and Fc fragments. FabALACTICA is specific for human IgG1 and digests at a specific site (…KSCDKT / HTCPPCP…) with overnight incubation without the need for reducing conditions. GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at a specific site (…KSCDK/THTCPPCP…).

Is GingisKHAN an IgG specific protease?

No, GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at one specific site (…KSCDK/THTCPPCP…) above the hinge, generating Fab and Fc.

How can I stop the GingisKHAN reaction?

The enzyme is a cysteine protease that can be inhibited by alkylating agents such as iodoacetamide. There are also several other known inhibitors to this enzyme, like KYT-36.

Can I use another reducing agent besides GingisKHAN reducing agent?

We recommend using the 10x reducing agent provided with GingisKHAN which has an optimized pH to provide full enzymatic activity without affecting the disulphide bridges between the light chain and Fd region of the heavy chain, keeping the Fab intact. If other reducing agent is used there is a risk for non-optimal reaction conditions. It is important to use freshly prepared Reducing Agent (max storage 6h) for guaranteed activity of GinigsKHAN.

Can you recommend a protocol for purification of the Fab?

Genovis provides a GingisKHAN Fab kit containing a specific Capture Select CH1 resin for further purification of the Fab. See this page for more information.

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