Chaperone‐assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli

Abstract

Adrenodoxin reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds. After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system. The purified protein was homogeneous enough for reproducible crystallization. The crystals diffracted X-rays isotropically beyond 1.7 A î resolution permitting a structure analysis.z 1999 Federation of European Biochemical Societies.

Cloning, Expression and Purification of Cindoxin, an Unusual Fmn‐Containing Cytochrome P450 Redox Partner

“…The cinB gene displays strong similarity (47 % similarity) to mitochondrial adrenodoxin reductase, the expression of which is greatly improved by the addition of the GroEL and GroES chaperones in E. coli. [13] Expression of pCW-CdR in 10 % ethanol, which induces chaperone expression in E. coli, [14] was unsuccessful. Direct co-overexpression of the construct pGRO12, which encodes the GroEL and GroES genes, with pCW-CdR again yielded only inactive, unfolded protein that was observed in the pellet from cell lysates.…”

The Structure of Adrenodoxin Reductase of Mitochondrial P 450 Systems: Electron Transfer for Steroid Biosynthesis

“…AdR was initially isolated from bovine adrenals and crystallized (Vonrhein et al, 1999), but there were not enough suitable crystals for solving the structure. Therefore, the corresponding cDNA was overexpressed in Escherichia coli and the recombinant AdR puri®ed to homogeneity (Vonrhein et al, 1999). Using the hanging drop vapor diffusion method, three interrelated crystal forms A, A H and A HH were obtained.…”

“…Crystal form A is related to A H and A HH . The unit cell of form A is four times larger than those of the others (Vonrhein et al, 1999). The mercury derivative was produced by soaking with 0.05 mM methylmercury acetate in buffer C with 12 % (w/v) PEG-8000 and 10 % (v/v) glycerol for about ten hours at 20 C.…”

Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli

“…This idea was first demonstrated by increasing the solubility of Rubisco, a large oligomeric protein, in cells that over-expressed GroEL and GroES (Goloubinoff et al, 1989). Subsequently, a number of increasingly elaborate plasmid systems were developed that permit co-expression of various combinations of chaperones, or other facilitators of protein folding to allow production of large amounts of proteins of interest (Perez-Perez et al, 1995; Nishihara et al, 1998; Vonrhein et al, 1999; Yanase et al, 2002; Stevens et al, 2003; Xu et al, 2005; Schlapschy et al, 2006; de Marco et al, 2007). Although over-expression of substrate-specific chaperone and co-chaperone combinations improves folding of some recombinant proteins, this sometimes results in bacterial toxicity and leads to decreased protein yield (Blum et al, 1992).…”