Expression, purification and characterization of recombinant Z alpha(1)-antitrypsin--the most common cause of alpha(1)-antitrypsin deficiency.
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.
Levina
Vita
V
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800, Australia.
Dai
Weiwen
W
Knaupp
Anja S
AS
Kaiserman
Dion
D
Pearce
Mary C
MC
Cabrita
Lisa D
LD
Bird
Phillip I
PI
Bottomley
Stephen P
SP
D019540:Area Under Curve; D004926:Escherichia coli / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin