α-actinin-1 is a cytoskeletal actin-binding protein found in stress fibers and focal adhesion sites, where it crosslinks actin filaments and attaches them to membrane junction complexes. Consequently, it plays an important role in cell migration. Its crosslinking function is thought to be negatively regulated by increased intracellular calcium levels. Upon binding of acalcium ion, calmodulin-like domain of human α-actinin-1 undergoes structural changes that also affect the dynamics of juxtaposed ABD domain. The exact mechanism of regulation and consequent conformational changes remain elusive. Therefore, we wish to characterise changes in the dynamics of ABD domain between calcium-free and calcium-bound formsof α-actinin-1 using NMR spectroscopy. Since α-actinin-1 is too large to be successfully analysed by NMR, we wish to prepare segmentally labeled protein where only ABD domain is isotopically labeled. Two separately expressed fragments could be ligated via protein trans-splicing using split inteins to form segmentally labeled protein. In this work we thus set the basis for the preparation of segmentally labeled α-actinin-1. We successfully isolated two intein constructs, each containing a fragment of split α-actinin-1 and ligated them via trans-splicing, using split intein Cfa. We also optimized the ligation of intein constructs and showed that the highest yield is obtained at temperature 33 °C. With a large-scale ligation of intein constructs we confirmed that the yield is high enough for the preparation of segmentally labeled α-actinin-1for NMR studies.
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