Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli

Abstract

The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity. After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70%. The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems. SLPI proved to be a potent inhibitor of translation in vitro. When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75%. The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA. metabolizing enzymes, such as RNase. In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61. Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin. On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA. Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPIVDNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E. coli. These results indicate that, in general, high-level expression of basic recombinant proteins in E. coli may be problematic.A number of recombinant proteins, such as human interferons (11, 19), rat insulin (2), and bacterio-opsin (5), are toxic to Escherichia coli when expressed in high amounts. Shortly after the induction of their synthesis, cell growth rates decline, and cultures stop growing. before reaching normal stationary phase. As a consequence of growth inhibition, the accumulation of product is suboptimal.Presumably, there are many mechanisms by which the expression of recombinant proteins in E. coli can interfere with essential metabolic processes and growth. For the secreted forms of human interferon (11) and insulin (2), toxicity is caused by the binding of the proteins to the inner membrane and disruption of protein export (6). Intracellularly produced recombinant proteins, such as methionylinsulin (2), can also be toxic to E. coli, but in this case the mechanism(s) leading to toxicity is unknown.We have encountered toxicity problems when the human protein secretory leukocyte protease inhibitor (SLPI) (16, 17) is expressed in E. coli. SLPI (Mr = 11,726) is a highly basic protein that is composed of two partially homologous domains that are internally cross-linked via sulfhydryl bonds (8,17). SLPI is a potent inhibitor of serine proteases and as an inhibitor of human leukocyte elastase may be of therapeutic value in the treatment of emphysema (17).As...

Comparative loss of activity of recombinant secretory leukoprotease inhibitor and alpha 1-protease inhibitor caused by different forms of oxidative stress

“…In all applied test systems, the loss of anti-NE activity was similar for rSLPI and α 1 -PI. This is not surprising, since the active centres of both SLPI (Met 73 ) [14] and α 1 -PI (Met 358 ) [1] carry methionine residues. It has clearly been demonstrated by other authors that with α 1 -PI, oxidation of this methionine residue induces a dramatic loss of activity [3,39].…”

“…Thompson (Synergen, Boulder, CO, USA); its synthesis has been described elsewhere [14][15][16]. The α 1 -PI used was a highly purified preparation obtained from human plasma (ART Biochemicals, Athens, GA, USA).…”

Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor.

“…The study population included 17 normal, nonsmoking individuals (9 female, 8 male, age 37±3 yr) (All data are presented as mean±standard error of the mean; all statistical comparisons were made using the two-tailed Student's t test). All had normal lung function, chest radiographs, and no history of lung disease.…”

“…In the SLPI ELISA (15), recombinant SLPI (rSLPI) identical to the naturally occurring SLPI, served as standard. The rSLPI was produced by Escherchia coli under the direction of a synthetic SLPI cDNA (16,17). The cells expressing rSLPI were resuspended and passed through a pressure homogenizer and the lysate centrifuged and applied to an ion exchanger.…”

Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis

“…For example, basic proteins can inhibit transcription or translation by binding to the nucleic acids (8,15), exported proteins can jam the cellular export machinery (13,19), and strong promoters (17) or long palindromes (9) can prevent plasmid replication. Although such cloning problems are common, they are rarely analyzed in detail.…”