Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae
Open accessAbstract
The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria–Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5mM nitrite or at least 10mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be −25mV and −175mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.
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The N-terminus of NapB encodes a hydrophobic signal sequence, roughly 30 amino acids long, which targets unfolded NapB for periplasmic export via the sec-dependent pathway.
“The N-terminus encodes a hydrophobic signal sequence, roughly 30 amino acids long, which targets the unfolded NapB for periplasmic export via the sec dependent pathway.186,252,253”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [252]
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Nitrate and periplasmic nitrate reductases
Sparacino-Watkins, C., Stolz, J. F., & Basu, P. (2014) · Chem. Soc. Rev. · doi:10.1039/c3cs60249d · Cited by 426 · see citing works
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- Authors
- BRIGÉ, A., COLE, J. A., HAGEN, W. R., GUISEZ, Y., & Van BEEUMEN, J. J.
- Affiliations
- Ghent University (BE)
- University of Birmingham (GB)
- Delft University of Technology (NL)
- Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Gent 9000, Belgium
- Corresponding author
- GUISEZ, Yves
- Venue
- Biochemical Journal
- Publisher
- Portland Press Ltd.
- Published
- 2001-06-08
- Volume
- 356
- Issue
- 3
- Pages
- 851-858
- ISSN
- 0264-6021 (ISSN-L)
- 1470-8728
- PMC
- PMC1221913
- OpenAlex
- W2095930892
- Cited by
- 23
- References
- 46
- Citations by year
- 2024: 1 · 2022: 1 · 2021: 1 · 2020: 1 · 2018: 1 · 2016: 1 · 2015: 1 · 2013: 1
- FWCI
- 1.84
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Biochemical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Ghent University Academic Bibliography (Ghent University) (submittedVersion)
- University of Birmingham Research Portal (University of Birmingham) (submittedVersion)
- Funders
- Vlaamse regering
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
- Keywords
- Haemophilus influenzae, nitrate reductase, recombinant protein overexpression, protein purification, signal peptide cleavage, leader peptidase, cytochrome c maturation, periplasmic secretion, N-terminal truncation
- MeSH terms
- Bacterial Proteins, Base Sequence, Electrophoresis, Polyacrylamide Gel, Haemophilus influenzae, Oxidation-Reduction, Recombinant Proteins, DNA Primers
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