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 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.
journal_name
J Bacterioljournal_title
Journal of bacteriologyauthors
Miller KW,Evans RJ,Eisenberg SP,Thompson RCdoi
10.1128/jb.171.4.2166-2172.1989subject
Has Abstractpub_date
1989-04-01 00:00:00pages
2166-72issue
4eissn
0021-9193issn
1098-5530journal_volume
171pub_type
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journal_title:Journal of bacteriology
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pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1128/JB.134.2.375-380.1978
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1128/jb.177.12.3587-3588.1995
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:1997-12-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.110.2.570-577.1972
更新日期:1972-05-01 00:00:00
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pub_type: 杂志文章
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更新日期:1985-07-01 00:00:00
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journal_title:Journal of bacteriology
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更新日期:1993-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.90.4.857-862.1965
更新日期:1965-10-01 00:00:00
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pub_type: 杂志文章
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更新日期:1978-04-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:1998-12-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/JB.120.3.1227-1237.1974
更新日期:1974-12-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:2009-07-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:2008-01-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.171.4.2042-2048.1989
更新日期:1989-04-01 00:00:00
abstract::Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Azotobacter vinelandii, like the corresponding enzyme from other organisms, is activated by acetyl coenzyme A and inhibited by l-aspartate. Both modifiers affect primarily the affinity of the enzyme for phosphoenolpyruvate. This is the first enzyme with a strictly ana...
journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.106.1.31-36.1971
更新日期:1971-04-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.171.7.4031-4037.1989
更新日期:1989-07-01 00:00:00