molecular weight markers  (Bio-Rad)

Journal: Frontiers in Microbiology

Article Title: The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III

doi: 10.3389/fmicb.2024.1378989

Figure Lengend Snippet: (A) SDS gel of the production and purification of P. megaterium YptQ (CgoN). Recombinant P. megaterium YptQ (CgoN) was produced in E. coli BL 21 as outlined in Material and Methods. The image shows an InstantBlue™ stained 12% SDS polyacrylamide gel after electrophoresis. Pierce™ unstained molecular weight marker protein ranging M r from 14,000 to 116,000 are shown in lane 1. Further, total cellular extracts before IPTG induction (lane 2); 2 h (lane 3) and 20 h (lane 4) after the addition of 200 μM IPTG-are next; followed by the soluble (lane 5) and insoluble fraction of the extract (lane 6) after ultracentrifugation. The flow through of the glutathione agarose (lane 7), washing fraction 1 (lane 8); washing fraction 4 (lane 9), elution fraction 1 (lane 10) and fraction 2 (lane 11) after PreScission™ protease cleavage, and the final GST-tag elution fraction (lane 12) are concluding the analysis. Fraction 10 contains a single YptQ (CgoN) band with a M r of 30,000 ± 3,000, nicely corresponding to the calculated molecular weight of the protein of 30,781 Da. (B) Native molecular weight and oligomerization state of P. megaterium YptQ determined via gel permeation chromatography. Analytical gel permeation chromatography of freshly purified recombinant P. megaterium YtpQ (CgoN) was performed using a 24 mL Superdex® 200 10/30 column on an Äkta purifier system under anaerobic conditions. Protein elution was followed by absorbance measurements at 280 nm. The column was equilibrated using the Gel Filtration Molecular Weight Markers Kit MWGF200 (Sigma Aldrich, Germany) composed of cytochrome c ( M r = 12,400), carbonic anhydrase ( M r = 29,000), bovine serum albumin ( M r = 66,000), alcohol dehydrogenase ( M r = 150,000), β-amylase ( M r = 200,000), and apoferritin ( M r = 400,000). Their elution position is given in M r (x1,000) on the top of the figure. The chromatogram shows one major peak of M r = 35,000 ± 5,000, indicating a monomeric YtpQ (CgoN) protein. (C) Time-resolved coproporphyrinogen III to coproporphyrin III conversion by P. megaterium YtpQ in the presence of FAD and menadione. Standard anaerobic YtpQ (CgoN) assays including 1 μM purified, recombinant YtpQ (CgoN), 10 μM coproporphyrinogen III, 3 μM FAD and 5 μM menadione were incubated at 30°C and 200 rpm. Samples were taken at different time points and analyzed by fluorescence spectroscopy with an excitation wavelength of 409 nm and emission measurements from 510 nm to 690 nm at 0 min (cyan), 5 min (turquoise), 10 min (yellow), 20 min (orange), 30 min (light blue), 45 min (dark blue), and 60 min (brown). (D) Kinetic analysis of coproporphyrinogen III oxidase activity of purified, recombinant P. megaterium YtpQ (CgoN). Anaerobic standard activity assays were conducted with 1 μM purified YtpQ (CgoN), 3 μM FAD and 5 μM menadione, and 1–20 μM of the substrate coproporphyrinogen III. A graphic representation of the initial velocity v 0 of coproporphyrin III formation by the enzyme as a function of substrate concentration is shown. A K M value of 3.95 μmol/L and a k cat of 0.63 min −1 were deduced.

Article Snippet: A Superdex® 200 HR 10/30 column run on an Äkta purifier system (GE Healthcare™ GmbH, Freiburg, Germany) was equilibrated under anaerobic condition in an anaerobic chamber with protein buffer I and calibrated using the Gel Filtration Molecular Weight Markers Kit MWGF200 (Sigma Aldrich, Chemie, Taufkirchen, Germany) composed of cytochrome c ( M r = 12,400), carbonic anhydrase ( M r = 29,000), bovine serum albumin ( M r = 66,000), alcohol dehydrogenase ( M r = 150,000), β-amylase ( M r = 200,000), and apoferritin ( M r = 400,000) according to the manufacturer’s instructions.

Techniques: SDS-Gel, Purification, Recombinant, Produced, Staining, Electrophoresis, Molecular Weight, Marker, GPC Assay, Filtration, Incubation, Fluorescence, Spectroscopy, Activity Assay, Concentration Assay