Expression of functionally active cytochrome b5 in Escherichia coli: isolation, purification, and use of the immobilized recombinant heme protein for affinity chromatography of electron-transfer proteins.
Cytochrome b5 is an integral membrane protein which is localized in endoplasmic reticulum membranes. In this paper we present the results on expression in E. coli, purification, and characterization of recombinant rat cytochrome b5. The full-length cDNA for rat liver microsomal cytochrome b5 has been modified using polymerase chain reaction (PCR) to introduce corresponding restriction sites as well as to insert silent mutations in the N-terminal sequence to increase the content of A and T nucleotides that prevents formation of elements of secondary structure of the mRNA transcripts and facilitates high expression. The expression plasmid was constructed by cloning of amplified cDNA to pCWori+ plasmid and used for transformation of E. coli DH5 alpha. The optimization of recombinant cytochrome b5 expression procedure induces expression level up to 3000 nmoles per liter of growth medium; this confers in the cells a deep pink color. The most interesting fact is that cytochrome b5 is expressed in this system in the reduced state. Recombinant cytochrome b5 was purified from solubilized cell membranes by a combination of ion-exchange chromatography and gel filtration. During purification, part of the cytochrome b5 is subjected to limited proteolysis with formation of truncated form. Sequencing of the N-terminal part of the recombinant cytochrome b5 indicates that it coincides with the sequence of rat cytochrome b5. Recombinant cytochrome b5 was found to have physicochemical, catalytic, and immunochemical properties to that of the native protein and was used as an efficient affinity matrix for purification of the various electron-transfer proteins.
Chudaev
M V
MV
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, ul.
Usanov
S A
SA
D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017421:Sequence Analysis; D013053:Spectrophotometry
D008565:Membrane Proteins; D011994:Recombinant Proteins; D000622:Aminolevulinic Acid; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System