Abstract
BackgroundBiliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. Biliverdin IXα, through interaction with biliverdin reductase, also initiates signaling pathways leading to anti-inflammatory responses and suppression of cellular pro-inflammatory events. The use of biliverdin IXα as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials. To address these limitations, methods to produce, recover and purify biliverdin IXα from bacterial cultures of Escherichia coli were investigated and developed.ResultsRecombinant E. coli strains BL21(HO1) and BL21(mHO1) expressing cyanobacterial heme oxygenase gene ho1 and a sequence modified version (mho1) optimized for E. coli expression, respectively, were constructed and shown to produce biliverdin IXα in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXα than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXα production by strain BL21(mHO1) (up to an average of 23. 5mg L-1 culture) in fed-batch mode and production by strain BL21 (HO1) in batch-mode was scalable to 100L bioreactor culture volumes. Synthesis of the modified ho1 gene protein product was determined, and identity of the enzyme reaction product as biliverdin IXα was confirmed by spectroscopic and chromatographic analyses and its ability to serve as a substrate for human biliverdin reductase A.ConclusionsMethods for the scalable production, recovery, and purification of biliverdin IXα by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXα and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.
Highlights
Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase
Bilirubin IXα is known to associate with cell membranes where it quenches the propagation of reactive oxygen species (ROS) [2,3] conferring protection to membrane lipids and proteins against oxidative damage
Though bilirubin IXα is an effective ROS quencher, biliverdin administered at tissue injury/ inflammatory sites appears as effective a cytoprotectant as bilirubin IXα [8,9,10,11,12,13]
Summary
Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. The use of biliverdin IXα as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials To address these limitations, methods to produce, recover and purify biliverdin IXα from bacterial cultures of Escherichia coli were investigated and developed. An additional function of biliverdin is to serve as the immediate source of bilirubin IXα that in turn acts as a cytoprotective antioxidant It is not clear if biliverdin is oxidatively regenerated after bilirubin IXα reacts with ROS [4,5,6,7]. The growing list of potential clinical applications for biliverdin suggests a future need for high-quality preparations in ample quantity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
