Format

Send to

Choose Destination
Protein Expr Purif. 2017 Jun;134:47-62. doi: 10.1016/j.pep.2017.03.019. Epub 2017 Mar 23.

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization.

Author information

1
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30239 Krakow, Poland. Electronic address: nceniedz@cyf-kr.edu.pl.
2
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30239 Krakow, Poland.
3
Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow 30060, Poland; Department of Molecular Physiology and Biological Physics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
4
Faculty of Agriculture and Economics, University of Agriculture in Krakow, Mickiewicza 21, 31120 Krakow, Poland.
5
Molecular and Environmental Sciences Group, Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada.
6
Department of Molecular Physiology and Biological Physics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
7
Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow 30060, Poland.

Abstract

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

KEYWORDS:

Chaperone protein; Molybdenum cofactor; Molybdoenzymes; Thermofluor shift assay

PMID:
28343996
PMCID:
PMC5535313
DOI:
10.1016/j.pep.2017.03.019
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center