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To be able to screen for potent and selective inhibitors sufficient amounts of protein are required. Here, we present an easy and efficient protocol for the recombinant expression of soluble and active murine cathepsin B and L. For this, we used the strain <jats:italic>E. coli<\/jats:italic> SHuffle\u00ae T7 Express which is capable of forming disulfide bridges in the cytoplasm. The enzymes were purified by immobilized nickel ion-affinity chromatography. Using different constructs and media, expression levels were significantly improved and expression yields of 80\u2009\u00b1\u20092\u00a0mg\u00a0L<jats:sup>\u22121<\/jats:sup> for procathepsin B, which is 16-fold better than previously reported expression yields for procathepsin B, and 37\u2009\u00b1\u20092\u00a0mg L<jats:sup>\u22121<\/jats:sup> for procathepsin L, were achieved. After activation with dithiothreitol at slightly acidic pH, in vitro kinetic parameters of both cathepsins were determined using the commonly used synthetic substrates Arg-Arg-AMC or Phe-Arg-AMC. Moreover, to investigate the impact of the short C-terminal propeptide of procathepsin B, it was deleted by site-directed mutagenesis, the shortened target protein was expressed and purified, activated in vitro, and its activity was similar to the variant bearing this C-terminal propeptide.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Key points<\/jats:title>\n            <jats:p>\n              <jats:italic>\u2022 Recombinant gene expression of cathepsin B and L in E. coli SHuffle\u00ae T7\u00a0Express<\/jats:italic>\n            <\/jats:p>\n            <jats:p>\n              <jats:italic>\u2022 Soluble cathepsin expression with high expression yields<\/jats:italic>\n            <\/jats:p>\n            <jats:p>\n              <jats:italic>\u2022 Investigation of the short C-terminal propeptide of cathepsin B<\/jats:italic>\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1007\/s00253-024-13374-1","type":"journal-article","created":{"date-parts":[[2024,12,16]],"date-time":"2024-12-16T10:53:31Z","timestamp":1734346411000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Improved recombinant expression of soluble cathepsin B and L in Escherichia coli"],"prefix":"10.1007","volume":"108","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8286-1589","authenticated-orcid":false,"given":"Christina","family":"M\u00f6ller","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4057-2057","authenticated-orcid":false,"given":"Niklas","family":"Rimkus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7028-3427","authenticated-orcid":false,"given":"Ferdinand F. 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