Physiological effects of producing plasminogen activator inhibitor-1 (PAI-1) in Escherichia coli
The effect of producing human plasminogen activator inhibitor-1 (PAI-1) on the physiology of the bacterium Escherichia coli has been investigated. PAI-1 belongs to the family of serine protease inhibitors. These inhibitors are distinct from others in that they contain the active site bond located on a mobile loop. A plasmid containing the PAI-1 gene was used to accumulate PAI-1 to 20% of the total cell protein. At least 50% of PAI-1 was accumulated as the active soluble protein. PAI-1 production has been shown to decrease the growth rate from 0.76 ± 0.038 h-1 (n = 4) to 0.54±0.038 h-1 (n = 4). Plasmids of similar construction producing either tumour necrosis factor α (TNFα) or an α2-interferon fragment [amino acids 4-155; IFN4-155)] caused no decrease in the growth rate. RpoS, an alternative sigma factor, was quantified by Western blotting. Cells producing PAI-1 had higher levels of RpoS than other recombinants; this was observed in both the exponential and stationary growth phases. These high levels of RpoS may be caused by the inhibition of the serine protease ClpP, or other proteases, by PAI-1. There is an accumulation of RpoS in the absence of ClpP as this protease is needed for the degradation of RpoS. No conclusive biochemical evidence was obtained to support this hypothesis. A correlation was found between possession of an intact rpoS gene and the specific ability to express PAI-1. N-ethylmaleimide (NEM) is a toxic electrophile to which E. coli cells are sensitive. On entry into the stationary phase, cells increase their expression of RpoS-dependent genes which confer resistance to NEM. The sensitivity of exponentially-growing cells to NEM is influenced by the growth rate. RpoS is more abundant at slow growth rates resulting in increased resistance to NEM. The strain producing PAI-1 was more sensitive to NEM during the exponential phase.