Scientists tweak proteins with ‘Tub-tag’

Published: 30-Sep-2015

The new method makes use of the enzyme tubulin-tyrosine ligase (TTL) and its target sequence


Researchers from Ludwig-Maximilians-Universitaet (LMU) in Munich, together with colleagues in Berlin, have developed a rapid and efficient technique for targeted chemo-enzymatic functionalisation of proteins with many potential therapeutic applications.

Selective intermolecular recognition is at the heart of all biological processes. Thus proteins that bind specifically to complementary chemical structures are also indispensable for many biochemical and biotechnological applications. Targeted modification of such proteins therefore plays a significant role in medical diagnostics and therapies.

The team, led by Professor Heinrich Leonhardt at LMU’s Biocentre and Professor Christian Hackenberger of the Leibniz Institute for Molecular Pharmacology in Berlin, have developed a new strategy that permits specific chemical modification of virtually any protein more rapidly and more efficiently than was hitherto possible. Their results are published in the journal Angewandte Chemie.

Many of the methods routinely used in the biosciences are based on the specific modification of proteins, particularly antibodies, to endow them with novel properties for specific purposes. For example, chemotherapeutic agents are often chemically linked to antibodies that recognise antigens found only on the surface of the target tumour. In this way, the cytoxic drug can be delivered directly to the cells it is intended to eradicate. Ideally, the methods used to introduce such modifications should be as specific, efficient and versatile as possible.

The ‘Tub-tag’ technology is characterised by extremely high efficiency and tremendous chemical flexibility

'Thanks to the combination of biotechnological and chemical expertise available for this co-operative project, we have succeeded in developing what we call the ‘Tub-tag’ technology, which is characterised by extremely high efficiency and tremendous chemical flexibility and is simple to perform,' says Hackenberger.

The new method is said to be the first to make use of the enzyme tubulin-tyrosine ligase (TTL) and its target sequence. TTL binds to a short amino-acid sequence found in its natural target – the cytoskeletal protein tubulin – and adds the amino-acid tyrosine to its C-terminal end. The researchers therefore refer to this guide sequence as the 'Tub-tag’.

'Our idea was to integrate this Tub-tag sequence into other proteins, thus turning them into targets for TTL. We have demonstrated the feasibility of this approach by introducing the Tub-tag into various nanobodies, which are downsized and stable derivatives of antibodies that we have used with great success in our laboratory for many years,' Leonhardt explains.

Since the engineered nanobodies are now recognised as targets by TTL, the enzyme can also be used to attach synthetic derivatives of tyrosine to them.

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