[Association of cytochrome P450 2B4 with molecular chaperones in heterological expression in E coli].
To produce a water-soluble form of microsomal P450 2B4, fusion proteins with glutathione-S-transpherase were genetically engineered. Specific proteolitic sites recognized 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 this site 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 specific proteinase action. Positive result has been obtained at specific proteolysis with IgA endoproteinase, recognizing the native sequence PPGP (31-34) in P450 2B4. Thus, at first time truncated form of cytochrome 2B4, lacking its 33 N-terminal amino acid residues has been created. It was found that the expression of genetically engineered variants of GST-2B4 in Escherichia coli is accompanied by tight complex formation with molecular chaperones 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 part of 2B4, and also before N-terminal methionine 2B4 and at position 33-34 (2B4). These results suggest the possibility that interaction with a GroEL/DnaK molecular chaperones may be requirement for correct folding of eukaryotic cytochrome 2B4 during its biosynthesis in E. coli.
Prozorovskiĭ
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TV
Orekhovich Institute of Biomedical Chemistry RAMS, Pogodinskaya 10, Moscow, 119992. proz@medic.ibmh.msk.su
Schunck
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WH
Archakov
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D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; C063842:dnaK protein, E coli