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Large‐scale expression and purification of active pseudolysin in Escherichia coli
Author(s) -
Tarpley Reid S.,
Adekoya Olayiwola A,
Sylte Ingebrigt,
Odunuga Odutayo O
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.984.4
Subject(s) - escherichia coli , maltose binding protein , proteases , fusion protein , protease , heterologous expression , biology , proteolysis , biochemistry , expression vector , microbiology and biotechnology , heterologous , chemistry , recombinant dna , enzyme , gene
Pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, is a member of the thermolysin‐like family of proteases. Pseudolysin has been identified as a key virulence factor in pathogenesis of P. aeruginosa infections due to its proteolytic activity on host tissue proteins causing severe hemorrhagic activity and weakening of the host defense mechanisms. The strong involvement of metalloendopeptidases in various diseases and infections makes them robust drug targets for therapeutic applications. Previous attempts to purify pseudolysin directly from P. aeruginosa or by heterologous expression in Escherichia coli and yeast yielded insufficient quantities of the enzyme for structural studies. We report the successful large‐scale heterologous expression of a correctly folded and active 33‐kDa pseudolysin in E. coli. The open reading frame coding for the secreted posttranslationally modified 33‐kDa pseudolysin was cloned into a plasmid vector and the protein product expressed in fusion with maltose‐binding protein (MBP). The MBP‐pseudolysin fusion protein expressed highly in E. coli and was found to be sufficiently soluble. Untagged pseudolysin obtained after cleaving the fusion protein with tobacco etch virus protease was soluble, stable and found to be active based on its ability to hydrolyze the Gly‐Leu bond in the substrate, 2‐aminobenzoyl‐Ala‐Gly‐Leu‐Ala‐4‐nitrobenzylamide.