Preprint
An engineered biofactory for efficient production of diverse recombinant superoxide dismutase isozymes loaded with specific metal ions for biochemical characterisation
bioRxiv
Cold Spring Harbor Laboratory
07/09/2026
DOI: 10.64898/2026.07.08.737244
PMID: 42465305
Abstract
Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside
cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (
MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of
lacking the endogenous SodFMs. Here, we characterised this
BL21 (DE3) Δ
Δ
strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for
characterisation and use.
Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein.
The
strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.
Details
- Title: Subtitle
- An engineered biofactory for efficient production of diverse recombinant superoxide dismutase isozymes loaded with specific metal ions for biochemical characterisation
- Creators
- Rafał Mazgaj - Institute of Biochemistry and Biophysics, Polish Academy of SciencesAgnieszka Kołpa - Institute of Biochemistry and Biophysics, Polish Academy of SciencesMariam Esmaeeli - Institute of Biochemistry and Biophysics, Polish Academy of SciencesJustyna Pełczyńska - Institute of Biochemistry and Biophysics, Polish Academy of SciencesDiana Galea - Institute of Biochemistry and Biophysics, Polish Academy of SciencesJan Gawor - Institute of Biochemistry and Biophysics, Polish Academy of SciencesAgata Malinowska - Institute of Biochemistry and Biophysics, Polish Academy of SciencesAgnieszka Szczypiorowska - Institute of Biochemistry and Biophysics, Polish Academy of SciencesThomas Kehl-Fie - University of IowaKevin J Waldron - Institute of Biochemistry and Biophysics, Polish Academy of Sciences
- Resource Type
- Preprint
- Publication Details
- bioRxiv
- DOI
- 10.64898/2026.07.08.737244
- PMID
- 42465305
- ISSN
- 2692-8205
- eISSN
- 2692-8205
- Publisher
- Cold Spring Harbor Laboratory; United States
- Language
- English
- Date posted
- 07/09/2026
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9985183395902771
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