| Uroporphyrinogen III methyltransferase (UMT), a key enzyme in the branched pathway in the biosynthesis of heme and siroheme, is a novel reporter owing to the catalytic products accumulated in cells emitting red florescence under UV light. Comparing with green fluorescent protein (GFP), application of UMT is still in infancy. In this study, we detected the expression level of UMT or UMT fused proteins under different conditions and applied UMT reporter for monitoring protein expression in Escherichia coli. The results are as follows:1. The function of the wild type and the evolved barely UMT in E. coli was analyzed. The cells expressing engineered variant displayed stronger fluorescence than those expressing wild type enzyme. The main pigments accumulated in the cells were trimethylpyrrocorphin and sirohydrochlorin by UV-visible and fluorescence scanning analyses.2. The effects of different E. coli strains, different promoters and induction conditions independently on two barley UMT constructs were investigated. Under all conditions tested, the cells producing the recombinant variant exhibited stronger fluorescence signal than those producing the wild type enzyme.3. Two peptide tags as the fusion partners on expression of both constructed enzymes were examined. The variant fused with a ssrA tag at C-terminus was degraded by the E. coli chromosome encoded protease C1pXP, whereas the wild type enzyme was expressed as inclusion bodies. Fusion of the N-terminal pleB peptide leader resulted in both proteins exported to the periplasm, but the fluorescence signals were decreased, as comparison to the enzymes expressed in the cytoplasm.4. Coexpression of molecular chaperones, additional pre-substrate 5-aminolevulinate, and different E. coli strains affecting on soluble expression level for maize UMT as the fusion partner were identified. The protein solubility of the wild type and mutated MBPs or tobacco etch virus protease constructs fused independently with the UMT reporter was not obviously influenced by the coexpressed chaperones and rare tRNA abundance. The exogenous 5-aminolevulinate disturbed the fluorescence of cells expressing the fusion proteins for three versions of the protease.5. The differently soluble expression level of the fusion protein for UMT was monitored. Corresponded with GFP and a-peptide, UMT can monitor solubility of three MBP or TEVp constructs. The low expression levels of some maize proteins in E. coli were detected using the UMT reporter, as well as GFP reporter. The N-terminal SUMO tag had the limited enhancing production of the fusion proteins.6. The coupled selection of protein solubility based on potassium tellurite inhibiting cell growth and fluorescence display were developed. With increasing concentrations of potassium tellurite, colony numbers of the mixed cells expressing the selected six proteins with different solubility were decreased, but colonies displaying red fluorescence was identified to be produced the expected protein with relatively high solubility by SDS-PAGE and Western blot analysis.7. The selected five enzymes involved in heme and siroheme biosynthesis impacting on UMT reporter were confirmed. The new fluorescent material from cells expressing the fusion protein for 5-aminolevulinate synthase or uroporphyrinogen decarboxylase was discovered preliminary by UV-visible scanning analysis. Other three enzymes had not significant effect on the change of cell fluorescence.Taken together, the different plant UMT proteins as the N-or C-terminal fusion partner are applied for monitoring the functional and soluble expression levels in E. coli. The limits of its application are also identified. Our developed reporter system will provide the efficient selection of protein solubility in E. coli. |
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