Alpha1-antitrypsin is the archetypal member of the serine protease inhibitor (serpin) superfamily. It is synthesised and secreted in the liver and its prime function is to form complexes with and inhibit the enzyme neutrophil elastase. This enzyme, released by neutrophils, is able to digest elastin fibres in the lungs and if uncontrolled may give rise to the destructive lung disease emphysema. A number of variant α1-antitrypsin molecules have been characterised which contain clinically relevant mutations. Substitution of the P1 methionine residue (position 358) in the reactive centre loop changes the inhibitory specificity of the molecule and may reduce or enhance its capacity to inhibit neutrophil elastase. The Z mutation (342 glutamic acid to lysine) results in loop sheet polymerisation of antitrypsin and accumulation of polymers within hepatocytes to give reduced circulating plasma levels. This accumulation of antitrypsin in the liver predisposes the individual to cirrhosis and, the low circulating plasma levels predispose to emphysema.
In an attempt to understand the effect these mutations have on the structure and function of the antitrypsin molecule, site directed mutants have been constructed in which the DNA coding for the P1 and P17 residues has been mutated to code for different amino acids. Mutants constructed include P1 arginine, leucine valine and P17 arginine, lysine, arginine, glutamine and histidine. These were then expressed as recombinant proteins in E.coli, using a 10 litre fermentation system, with a maximal biomass production of approximately 24g/l dry weight. The proteins were extracted from the cell mass, purified to homogeneity and partially characterised. Recovery of between 2 and 15 grams of recombinant antitrypsin per purification was obtained depending on the mutant expressed.[Show full abstract]
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