期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 68, 期 2, 页码 226-232出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2009.06.011
关键词
Serpin; Conformational disease; Proteinase inhibitor; Aggregation; Polymerization; Protein misfolding
类别
资金
- NHMRC
alpha(1)-Antitrypsin (alpha(1)AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. alpha(1)AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe alpha(1)AT deficiency are caused by the Z variant (E342K) of alpha(1)AT. The presence of the Z mutation results in misfolding and polymerization of alpha(1)AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z alpha(1)AT production. This has created a major impediment to studying the effect of the Z mutation on alpha(1)AT. Here we report our attempts to produce recombinant Z alpha(1)AT 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 alpha(1)AT. Cytosolic expression of the Z alpha(1)AT gene in P. pastoris was successful. Monomeric and active recombinant Z alpha(1)AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z alpha(1)AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z alpha(1)AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies. (C) 2009 Elsevier Inc. All rights reserved.
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