Two proposed mechanisms for 4-thiouridine generation share key cysteine persulfide and disulfide intermediates, and indirect evidence of their existence has been previously reported; chemical trapping and mass spectrometry have now provided direct and definitive evidence of these key intermediates.
The exquisite specificity of proteins is a key feature driving their application to anticancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown. Here, we describe a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice. We demonstrate this approach by creating a prodrug-activating enzyme that selectively kills human cancer cells that accumulate the marker hypoxia-inducible factor 1α. This property arises primarily through increased cellular accumulation of the enzyme in the presence of the marker. Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases. directed evolution | protein engineering | protein switch | enzyme/prodrug therapy
RG13 is a 72 kDa engineered allosteric enzyme comprised of a fusion between maltose binding protein (MBP) and TEM1 beta-lactamase (BLA) for which maltose is a positive effector of BLA activity. We have used NMR spectroscopy to acquire [(15)N, (1)H]-TROSY-HSQC spectra of RG13 in the presence and absence of maltose. The RG13 chemical shift data was compared to the published chemical shift data of MBP and BLA. The spectra are consistent with the expectation that the individual domain structures of RG13 are substantially conserved from MBP and BLA. Differences in the spectra are consistent with the fusion geometry of MBP and BLA and the maltose-dependent differences in the kinetics of RG13 enzyme activity. In particular, the spectra provide evidence for a maltose-dependent conformational change of a key active site glutamate involved in deacylation of the enzyme-substrate intermediate.
In support of the key features of sulfur transfer in the proposed mechanisms of 4-thiouridine generation, the enzyme ThiI can turn over only once in the absence of reductants of disulfide bonds, and Cys-456 of ThiI receives the sulfur transferred from the persulfide group of the sulfurtransferase IscS.
Bioinformatic analysis of endogenous CHO promoter sequences. Linking transcriptomic and genomic datasets for bioprocess-directed promoter design. In silico DOE-based design method for synthetic promoter construction. Design promoters exhibited 2.5-fold increase in activity over CMV-IE promoter. Promoters designed to function in context of a biphasic fed-batch production process.
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