Increased cytoplasmic expression of PETase enzymes in E. coli
We provide protein expression methods to produce three important PETase variants. Importantly, for IsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mg<sub>PETase</sub> g<sub>CDW</sub><sup>-1</sup> h<sup>-1</sup>, which is tenfold greater than that for K. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends to IsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes.
Authors
- Jason A. Berberich (ORCID: https://orcid.org/0000-0002-1396-6993)
- Joel L. Kaar (ORCID: https://orcid.org/0000-0002-0794-3955)
- Jason T. Boock (ORCID: https://orcid.org/0000-0002-5111-0969)
- L Carter
- T Sen (ORCID: https://orcid.org/0009-0008-7983-8282)
- Chris E. MacFarlane
- Samuel P. Karlock
Institutions
- University of Colorado Boulder (US)
- Miami University (US)
Publication Details
- Journal
- Microbial Cell Factories
- Published
- 2024-11-25
- DOI
- https://doi.org/10.1186/s12934-024-02585-w
- Citations
- 10
- Primary Topic
- Microplastics and Plastic Pollution
- Type
- article
- Field-Weighted Citation Impact
- 1.33
Funders
- National Science Foundation
- University of Miami
- Division of Chemical, Bioengineering, Environmental, and Transport Systems