Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation.

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.