Publication

Activity-independent screening of secreted proteins using split GFP

Knapp, A., Ripphahn, M., Volkenborn, K., Skoczinski, P. & Jaeger, K-E., 20-Sep-2017, In : Journal of Biotechnology. 258, p. 110-116 7 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • Andreas Knapp
  • Myriam Ripphahn
  • Kristina Volkenborn
  • Pia Skoczinski
  • Karl-Erich Jaeger

The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.

Original languageEnglish
Pages (from-to)110-116
Number of pages7
JournalJournal of Biotechnology
Volume258
Publication statusPublished - 20-Sep-2017
Externally publishedYes

    Keywords

  • Bacillus subtilis, Signal peptide screening, Split GFP, Lipase, Cutinase, Swollenin EXLX1, BACILLUS-SUBTILIS, BACTERIAL EXPANSIN, ESCHERICHIA-COLI, GENE-EXPRESSION, LIPOLYTIC ENZYME, FUSION, TRANSFORMATION, GENERATION, STABILITY, REPORTER

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