Split Inteins Without Active Site Cysteine
New route to protein-coupling via intein trans-splicing in the absence of reducing agents . Inteins are protein domains that auto-catalyze protein-splicing of their flanking regions with a peptide bond.In this invention novel cysteine-less split inteins are capable of robust trans-splicing at ambient temperatures and without any chemical reducing or denaturation.
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
The present invention allows for new strategies in the semisynthetic synthesis and site-selective bioconjugation of proteins and in biotechnology.
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 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.
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.
Licensing
14.06.2019
Patent application
5640
Germany
