Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli.
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 SLPI/DNA 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.
Miller
K W
KW
Human Pharmaceuticals Section, Synergen, Inc., Boulder, Colorado 80301.
Evans
R J
RJ
Eisenberg
S P
SP
Thompson
R C
RC
D001426:Bacterial Proteins / Q000096:biosynthesis; D002474:Cell-Free System; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects*; D009113:Muramidase / Q000378:metabolism; D011480:Protease Inhibitors / Q000633:toxicity*; D014176:Protein Biosynthesis / Q000187:drug effects; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000633:toxicity*; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000633:toxicity*; D012270:Ribosomes / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011480:Protease Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D009113:Muramidase