Expression of a biologically active plant cytochrome b5 in Escherichia coli.
Cytochrome b5 from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol. 185, 60-89). Transformed cells were red in colour and accumulated cytochrome b5 to a level of around 30% of the total cell protein. The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b5, and exhibited a c.d. spectrum resembling that of a mammalian cytochrome b5. The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus). Inhibition of haem synthesis in the transformed E. coli cells expressing cytochrome b5, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b5 holoprotein but had little effect on the accumulation of cytochrome apoprotein. The recombinant protein expressed in E. coli therefore has the biochemical features of the higher-plant cytochrome b5 and can be used in studies of plant microsomal oxidation/reduction reactions.
Smith
M A
MA
Department of Botany, University of Bristol, U.K.
Napier
J A
JA
Stymne
S
S
Tatham
A S
AS
Shewry
P R
PR
Stobart
A K
AK
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006418:Heme / Q000096:biosynthesis; D008322:Mammals; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves / Q000378:metabolism; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014026:Nicotiana / Q000378:metabolism*
D003509:Cyclohexanecarboxylic Acids; D017931:DNA Primers; D011994:Recombinant Proteins; C012987:gabaculine; D006418:Heme; D015786:Cytochromes b5