Expression, purification and characterization of recombinant Z α_1-Antitrypsin-The most common cause of α_1-Antitrypsin deficiency

摘要

α_1-Antitrypsin (α_1AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. α_1AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe α_1AT deficiency are caused by the Z variant (E342K) of α_1AT. The presence of the Z mutation results in misfolding and polymerization of α_1AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z α_1AT production. This has created a major impediment to studying the effect of the Z mutation on α_1AT. Here we report our attempts to produce recombinant Z α_1AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z α_1AT. Cytosolic expression of the Z α_1AT gene in P. pastoris was successful. Monomeric and active recombinant Z α_1AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z α_1AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z α_1AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.