title: Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation.
abstract: Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies. alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E. coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form. Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein. In addition, this variant proved to be fully functional as a proteinase inhibitor. Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: A J
authors.initials: AJ
authors.lastname: Schulze
authors.forename: E
authors.initials: E
authors.lastname: Degryse
authors.forename: D
authors.initials: D
authors.lastname: Speck
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: R
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000515:alpha 1-Antitrypsin