New approaches for the site-selective chemical modification of therapeutic proteins under mild conditions

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The site-specific chemical modification of protein therapeutics, for example antibodies, protein hormones, and cytokines, is of great importance for applications in cancer and disease treatment, diagnostics, and imaging. Chemical modifications can endow the protein therapeutics with new and tailored properties, for example longer circulation time in the blood-stream, targeted cell-killing (immunoconjugates), and reporting their localization (e.g. by fluorophores or tracers). In this project, we explore protein trans-splicing catalyzed by split inteins. Split inteins ligate their fused peptide or protein sequences with a native peptide bond and concomitantly remove themselves during the reaction, thus making the approach virtually traceless. We have developed and continue to improve two types of approaches to use protein trans-splicing for the preparation of chemically modified proteins. The synthetic moiety is transferred to the target protein either by splicing of a synthetic peptide with a recombinant protein (semi-synthetic protein trans-splicing) or by splicing of a short tag prelabeled at a unique cysteine or unnatural amino acid residue (chemical-tag splicing). In the second funding period we will generate new functional, chemically modified proteins and analyze them for biochemical and biomedical properties. The focus will be on antibody fragments potentially useful as protein therapeutics and for cellular imaging. We will also continue to develop new split-intein based technologies for selective chemical modification of proteins under mild and native conditions. These protein chemical tools are expected to be of high interest for a broad range of applications.