Cloning of truncation variants of α1-antitrypsin

Introduction Alpha1-antitrypsin is a member of the serpin (serine protease inhibitor) family of proteins . It controls the breakdown of elastic tissue in the lung by inhibiting human neutrophil elastase. It does so by the remarkable serpin mechanism forming a covalent complex with the enzyme and undergoing a dramatic, stabilising conformational change in order to fully disable it . However this potential for structural rearrangement renders serpins vulnerable to point mutations in critical regions, allowing aberrant conformational change in the absence of target protease. Individual molecules in which this occurs selfassociate to form chains known as polymers. Serpin polymerisation results in both loss of normal function and gain of toxic function, and underlies a group of diseases known as the serpinopathies . Pathogenic mutations in α1-antitrypsin cause its polymerisation within hepatocytes, giving rise to liver disease, while deficiency of the functional protein predisposes to catastrophic lung damage (emphysema) . A number of techniques (biochemical, biophysical, crystallographic) have been used to understand the general principals of the conformational changes that lead to serpin polymerisation . This has allowed the development of compounds capable of blocking them in a process of structure led drug design . However optimising this process requires more detailed understanding of the formation of polymerogenic intermediates in solution. Nuclear magnetic resonance spectroscopy is an excellent technique for such studies, however studying fulllength α1-antitrypsin is challenging because of its relatively large size (42kDa). This may be addressed by the generation of complementary pairs of truncated α1-antitrypsin constructs that can be covalently linked together to form functional full-length protein. Since the different fragments can be produced in either labelling or non-labelling conditions this approach allows spectra from much shorter sections of the polypeptide chain to be visualised and analysed.