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Principal Investigator: Joseph Shiloach
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2020
Award: $280,498
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
The following are couple of examples the first is the Effect of restricted dissolved oxygen on expression of Clostridium difficile toxin A submit from E. coli. And the second is related to expression of protein from pichia pastoris
The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS combined repetitive oligopeptides) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.
Concerning the yeast pichia pastoris: By evaluating different induction strategies, it was possible to increase the expression of Pgp more than 1000-fold. This result was obtained by inducing the expression with 20% (v/v) media containing 2.5% (v/v) methanol. By quantifying the Pichia biomass, the concentrations of methanol, formaldehyde, hydrogen peroxide, formate, and the enzymatic activities of alcohol oxidase (AOX), catalase (CAT), formaldehyde dehydrogenase (FLD), formate dehydrogenase (FDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), and -ketoglutarate dehydrogenases (-KGDHs), it was possible to establish a correlation between Pgp expression and the induction strategy. Inducing the culture by adding methanol together with fresh media was associated with decreased of formaldehyde and hydrogen peroxide and increased activities of FLD, FDH, IDH, and, -KGDHs. These results indicate that the lower Pgp expression was due to an increase in toxic formaldehyde and hydrogen peroxide and is not related to methanol oxidation. It is possible that Pgp expression is responsible for this behavior, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions, or when different proteins were expressed. The development of the optimized procedure is important for improving the production but understanding the effect of this specific protein on the host metabolism is equally important.
Terms: <ANK Domain><ANK Repeat><Aerobic><Affect><Anaerobic Bacteria><Ankyrin Repeat><Ankyrin Repeat Domain><Bacteria><Behavior><Binding><Biomass><C diff><C difficile><C-terminal><C. diff><C. difficile><CD enterotoxin><CD enterotoxin A><Carbinol><Cell Wall><Cellular Stress Response><Chaperone><Characteristics><Clinical><Clostridioides difficile><Clostridium difficile><Clostridium difficile enterotoxin A><Clostridium difficile tcdA protein><Clostridium difficile toxA protein><Clostridium difficile toxin A><DNA Sequence><Dehydrogenases><Development><Down-Regulation><Downregulation><E coli><E. coli><Escherichia coli><Formaldehyde><Formic Aldehyde><Generalized Growth><Genes><Growth><H2O2><Hansenula><Heat Shock><Heat-Shock Reaction><Heat-Shock Response><Hydrogen Peroxide><Hydroperoxide><Immunity><Intermediary Metabolism><Investigation><Isocitrate Dehydrogenase><Lead><Malate Dehydrogenase><Malic Dehydrogenase><Messenger RNA><Metabolic Processes><Metabolism><Methanol><Methyl Alcohol><Methyl Aldehyde><Molecular Chaperones><Molecular Interaction><NAD-Malate Dehydrogenase><Non-Polyadenylated RNA><O element><O2 element><Oligopeptides><Oxidoreductase><Oxidoreductase Gene><Oxomethane><Oxygen><Pathway interactions><Pb element><Physiologic><Physiological><Pichia><Procedures><Process><Production><Proteins><Proteome><RNA><RNA Gene Products><Recombinant Proteins><Recombinants><Reductases><Reporting><Ribonucleic Acid><Ribosomes><Stress><Structure><Time><Tissue Growth><Transcription Regulation><Transcription Repressor><Transcriptional Control><Transcriptional Regulation><Transcriptional Repressor><Translations><Up-Regulation><Upregulation><Vaccine Production><Vaccines><Wood Alcohol><Work><Yeasts><alcohol oxidase><alpha Toxin><anaerobe><base><catalase><density><developmental><differential expression><differentially expressed><genetic repressor><global gene expression><global transcription profile><heavy metal Pb><heavy metal lead><improved><ketoglutarate dehydrogenase><mRNA><microorganism><ontogeny><oxidation><pathogen><pathway><protein E><protein expression><protein folding><resistance strain><resistant strain><response><transcriptional differences><transcriptome><vaccine candidate><α-Toxin>