Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli.
Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.
Bischoff
R
R
Transgene S.A., Strasbourg, France.
Speck
D
D
Lepage
P
P
Delatre
L
L
Ledoux
C
C
Brown
S W
SW
Roitsch
C
C
D000595:Amino Acid Sequence; D001322:Autoanalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004731:Endotoxins / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D014644:Genetic Variation; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics*
D004731:Endotoxins; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin