ABSTRACT. Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy with no cure currently available. In this study, using two microarray data sets obtained from the Gene Expression Omnibus database, we conducted a dysfunctional pathway-enrichment analysis and investigated deregulated genes that are specific to different phases of the disease in order to determine pathogenic characteristics in the progression of DMD. We identified 41 and 33 dysfunctional pathways that were enriched with differentially expressed genes in presymptomatic patients and in symptomatic patients, respectively. Over 70% of pathways were shared between both phases and many of them involved the inflammatory process, suggesting that inflammatory cascades were induced soon after the birth of the patients. Further investigation showed that presymptomatic patients performed better with respect to muscle regeneration and cardiac muscle calcium homeostasis maintenance. Neuronal nitric oxide synthase, dihydropyridine receptors, sarcoplasmic/ endoplasmic reticulum calcium ATPase, and phospholamban may serve as potential targets for further molecular diagnostic tests. Our results may provide a better understanding for the treatment of DMD.
CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.
Background The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in Pichia pastoris. Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of P. pastoris are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable. Results The coexpression strains harboring different copies mtg were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of P. pastoris . The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of mtg gene ( mtg- 3c) was calculated according to the standard curve of gap and mtg genes ( gap is regarded as the single-copy reference gene). The maximum enzyme activity of mtg- 3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with mtg -2c, mtg -6c or mtg -8c, mtg -3c is the highest expression level and enzyme activity, implying that mtg -3c is the most suitable for co-expression pro-peptide and MTG. Conclusions This study provides an effective strategy for improving the expression level of active MTG by directional increasing of mtg copies in P. pastoris. Electronic supplementary material The online version of this article (10.1186/s12896-019-0542-6) contains supplementary material, which is available to authorized users.
BackgroundInterferon (IFN)-α has been commonly used as an antiviral drug worldwide; however, its short half-life in circulation due to its low molecular weight and sensitivity to proteases impacts its efficacy and patient compliance.ResultsIn this study, we present an IgG1 Fc fusion strategy to improve the circulation half-life of IFN-α. Three different forms of IgG1 Fc fragments, including the wild type, aglycosylated homodimer and aglycosylated single chain, were each fused with IFN-α and designated as IFN-α/Fc-WT, IFN-α/Fc-MD, and IFN-α/Fc-SC, respectively. The recombinant proteins were expressed in Pichia pastoris and tested using antiviral and pharmacokinetic assays in comparison with the commercial pegylated-IFN-α (PEG-IFN-α). The in vitro study demonstrated that IFN-α/Fc-SC has the highest antiviral activity, while IFN-α/Fc-WT and IFN-α/Fc-MD exhibited antiviral activities comparable to that of PEG-IFN-α. The in vivo pharmacokinetic assay showed that both IFN-α/Fc-WT and IFN-α/Fc-MD have a longer half-life than PEG-IFN-α in SD rats, but IFN-α/Fc-SC has the shortest half-life among them. Importantly, the circulating half-life of 68.3 h for IFN-α/Fc-MD was significantly longer than those of 38.2 h for IFN-α/Fc-WT and 22.2 h for PEG-IFN-α.ConclusionsThe results demonstrate that the elimination of N-glycosylation by mutation of putative N-glycosylation site further prolongs the half-life of the IFN-α/Fc fusion protein and could present an alternative strategy for extending the half-life of low-molecular-weight proteins expressed by P. pastoris for in vivo studies as well as for future clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0601-9) contains supplementary material, which is available to authorized users.
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