Recombinant equine cytochrome c in Escherichia coli: high-level expression, characterization, and folding and assembly mutants.
To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to the ribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variants tailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform.
Rumbley
Jon N
JN
The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, USA. jr@hxiris.med.upenn.edu
Hoang
Linh
L
Englander
S Walter
SW
D000818:Animals; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D019791:Guanidine; D006736:Horses; D007700:Kinetics; D016296:Mutagenesis; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D019791:Guanidine