ABSTRACT
One way of limiting the environmental impact of food production and improving food security is to replace part of the animal- or plant-based protein in the human diet with protein sourced from microorganisms. The recently discovered bacterium Xanthobacter sp. SoF1 (VTT-E-193585) grows autotrophically using carbon dioxide gas as the only carbon source, yielding protein-rich biomass that can be processed further into a powder and incorporated into various food products. Since the safety of this microbial protein powder for human consumption had not been previously assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found.
ABSTRACT
BACKGROUND: We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation. RESULTS: We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield. CONCLUSIONS: Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.
