A, Linear map of the pVP56K-fl-cytb5 expression vector. B, amino acid sequence of the His8-MBP-fl-cytb5 fusion protein, where fl indicates the full-length protein. The His8 tag, TEV protease cleavage site, and the first five residues the fl-cytb5 liberated by TEV proteolysis are shown in bold letters. The C-terminal membrane anchor domain is underlined. C, schematic of the fusion proteins studied here.

Difference in post-translational processing caused by long-term auto-induction or short-term IPTG induction. TEV proteolysis emphasizes the difference in size of the cytb5 variants. Lane 1, peak 1 from Ni IMAC purification of His8-MBP-fl-cytb5 expressed by auto-induction; cytb5 19 designates a truncated form lacking 19 residues from the C-terminus. Lane 2, peak 2 from Ni IMAC purification of His8-MBP-fl-cytb5 expressed by auto-induction. Lane 3, peak from Ni IMAC purification of His8-MBP-fl-cytb5 expressed by IPTG induction.

Steady-state reduction of His8-MBP-fl-cytb5 (○) and His8-cytb5-sd (●) by His8-cytb5R. The lines are least square fits to the simple Michaelis-Menten assumption.

Incorporation of fl-cytb5 into phospholipids vesicles. A, a schematic representation of the liposome floating experiment. The open circles represent the liposomes and the solid black circles represent proteins, including His8-MBP-fl-cytb5 and fl-cytb5 and TEV protease. Protein bound to the liposome will migrate to the interface between buffer and 30% Accudenz, while unbound proteins will remain in the 40% Accudenz layer. B, Alignment of scintillation counting results (detects [³H]-1,2-dipalmitoyl phosphatidylcholine added as a tracer to the liposome preparation) of the fractions collected from top to bottom from the liposome floating experiment with a Coomassie-stained denaturing gel. Lanes 1–3 contain the liposomes and fl-cytb5 (4). His8-MBP and TEV protease did not bind to liposomes. In control experiments (not shown), His8-MBP-fl-cytb5 was also bound to liposomes in the absence of TEV protease. Lanes 8–10 contain residual His8-MBP-fl-cytb5 (1), His8-MBP (2), TEV protease (3), and unbound fl-cytb5 (4). C, Alignment of scintillation counting results ([³H] lipid) with Coomassie-stained denaturing gel for exposure of cytb5-sd to liposomes. Cytb5-sd (5) did not bind to liposomes.

Coomassie-stained denaturing gel electrophoresis. 1, 100 μg of purified cytb5-sd, where sd indicates the soluble domain. 2, 100 μg of purified His8-cytb5R, where R indicates the reductase protein. 3, molecular weight markers. 4, total cell lysate from E. coli cells expressing His8-fl-cytb5, where fl indicates the full-length protein. 5, insoluble fraction. 6, soluble fraction. 7, molecular weight markers. 8, Soluble fraction from E. coli cells expressing His8-MBP-fl-cytb5. 9, His8-MBP-fl-cytb5 purified Ni IMAC chromatography.

Spectra changes in His8-MBP-fl-cytb5 during a time-course reduction with soluble His8-cytb5R in the presence of NADH. As assembled, the reaction went to completion in ~30 s. A blank was obtained with buffer solution containing NADH, and the spectral contribution of His8-cytb5R (1.3 nM) was negligible.

Coomassie-stained denaturing gel electrophoresis showing the time and concentration dependence of proteolysis of His8-MBP-fl-cytb5 in the presence of TEV protease to release fl-cytb5. A, the TEV protease was present at ratio of 1:10 relative to His8-MBP-fl-cytb5. (1) is His8-MBP-fl-cytb5, (2) is His8-MBP, (3) is TEV protease, and (4) is fl-cytb5. B, TEV protease to His8-MBP-fl-cytb5 ratio of 1:50. C, TEV protease to His8-MBP-fl-cytb5 ratio of 1:100.