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Split Inteins Without Active Site Cysteine


Abstract

New route to protein-coupling via intein trans-splicing in the absence of reducing agents


Background

Inteins are protein domains that auto-catalyze protein-splicing of their flanking regions with a peptide bond. In protein trans-splicing two parts of a split-intein can be used to ligate or cyclize polypeptides in a traceless manner. The reaction is robust, can be performed in-vivo or in-vitro, using biologically or chemically synthesized peptides or proteins. The sequence of only 2-3 flanking amino acids are constrained. As shown in the figure below, the N and C terminal intein fragments (IntN & IntC) associate and fold into the active domain thereby linking the flanking sequences with a peptide bond.


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Solution

All previously known split inteins only function in reducing conditions due to their use of one or two catalytic cysteines. In this invention novel cysteine-less split inteins are capable of robust trans-splicing at ambient temperatures and without the requirement of any chemical reducing or denaturation steps. This allows the preservation of disulfide bonds within the target protein.

The use of this split intein was demonstrated for a full-length IgG, an Fc fragment of an IgG antibody and two nanobodies as representatives of therapeutically relevant proteins. The reactions are high yielding (> 90%) at low to medium micromolar concentrations.


Advantages

  • Performed in absence of reducing agents
  • Reactions at ambient temperature; high yield
  • Novel access to protein semi synthesis and protein modification without denaturation
  • Preservation of disulfide bonds and free cysteines

PROvendis GmbH

Dr. Andreas Wagener
+49.208 94105-38
aw@provendis.info
www.provendis.info
Address
Schloßstr. 11-15
45468 Mülheim an der Ruhr



Development status

Proof of concept


Patent situation

  • DE pending
  • EP pending
  • US pending

Keywords

split inteins, cystein free

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