Overexpression of a Rhodococcus erythropolis protein in Escherichia coli with immunological identity to the Rhodococcus steroid 1‐dehydrogenase. Immunoelectron microscopic localization and electrophoretic studies

Abstract

The recombinant Escherichia coli K-12 strain chi 6060 harbouring the plasmid pYA 1201 with a gene from Rhodococcus erythropolis IMET 7030 overexpressed a protein which reacts with a monospecific antiserum against the steroid 1-dehydrogenase (Sdh) from the same Rhodococcus strain. It was shown previously that this recombinant protein exhibits no enzymatic activity. By immunogold labelling the protein was localized on ultrathin sections of the recombinant E. coli strain. After cultivation at 37 degrees C it was found within large cytoplasmic compartments (inclusion bodies). The inclusion bodies occupied 40% to 75% of the sectioned cell area. The highest amount of protein was observed after induction of the culture with isopropyl-beta-D-thiogalactopyranoside. Approximately 20% of the induced cells became enlarged (up to 5-fold of the normal size) and deformed; multiplication of the Rhodococcus protein producing cells was inhibited. After ultrasonic cell disintegration the inclusion bodies were found only in the fraction of the sedimented cell debries and did still react with anti-Sdh. When recombinant E. coli cells were cultivated at 28 degrees C, inclusion bodies appeared very seldom and the immunoreactive protein was distributed throughout the whole cytoplasm.

Localisation of the cell wall-associated phosphodiesterase PhoD ofBacillus subtilis

“…For DNA amplification a VENT Taq polymerase with proof‐reading activity (New England Biolabs, Inc.) was used. Anti‐rabbit IgG‐gold was prepared by adsorption of swine anti‐rabbit IgG (Seravac, Prague) to colloidal gold with a mean particle size of 16 nm [21] in borate buffer as described [22].…”

Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ 1 -Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

“…Only the KSTD-encoding genes of A. simplex, Comamonas testosteroni, and Rhodococcus rhodochrous have been fully characterized (23,24,30). Although cloning of the gene encoding KSTD and expression of an inactive KSTD protein of R. erythropolis IMET7030 in Escherichia coli have been described (2,38,39) and a nucleotide sequence of the KSTD-encoding gene of N. opaca (10) (synonym, R. erythropolis IMET7030 [15]) is available (DDBJ/EMBL/GenBank accession no. U59422), no further characterization of this gene has been reported.…”