Voprosy meditsinskoi khimii (ISSN 0042-8809)

Association of cytochrome P450 2B4 with molecular chaperones after hetrologousexpression in e.coli


1. Orekhovich Institute of Biomedical Chemistry RAMS
2. Max-Delbruck-Centre for Molecular Medicine, Berlin, Germany
PubMed Id: 11558314
Year: 2001 vol: 47  issue:3  pages: 315-328
Abstract: To produce a water-soluble form of microsomal P450 2B4, fusion proteins withglutathione-S-transpherase were genetically engineered. Specific proteolitic sitesrecognized by Factor Xa and Thrombin have been introduced into N-terminus of P450 2B4(46-49), lacking signal anchor sequence (2-27). It was supposed that proteolysis at thissite could give the possibility to produce protein lacking hydrophobic N-terminus sequence(1-49). However, it was shown that given region in P450 2B4 his resistant against specificproteinase action. Positive result has been obtained at specific proteolysis with IgAendoproteinase, recognizing the native sequence PPGP (31-34) in P450 2B4. Thus, at firsttime truncated form of cytochrome 2B4, lacking its 33 N-terminal amino acid residues hasbeen created. It was found that the expression of genetically engineered variants ofGST-2B4 in Escherichia coli is accompanied by tight complex formation with molecularchaperones GroEL and DnaK. Dissociation of the complex occurred after proteolysis in:linker sequence (position 6-7) between C-terminal part of GST domain and N-terminal partof 2B4, and also before N-terminal methionine 2B4 and at position 33-34 (2B4). Theseresults suggest the possibility that interaction with a GroEL /DnaK molecular chaperonesmay be requirement for correct folding of eukaryotic cytochrome 2B4 during itsbiosynthesis in E.coli.
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Reference: Prozorovsky T.V., Schunck W.H., Archakov A.I., Association of cytochrome P450 2B4 with molecular chaperones after hetrologousexpression in e.coli, Voprosy meditsinskoi khimii, 2001, vol: 47(3), 315-328.