ABSTRACT
Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.
Subject(s)
CRISPR-Cas Systems , Cricetulus , Gene Editing , CHO Cells , Animals , CRISPR-Cas Systems/genetics , Gene Editing/methods , Antibodies, Monoclonal/genetics , Recombinant Proteins/genetics , Gene Knockout Techniques/methods , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Cricetinae , Genetic Engineering/methodsABSTRACT
Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m-1s-1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.
Subject(s)
Bacterial Proteins/metabolism , Chorismate Mutase/metabolism , Mycobacterium tuberculosis/enzymology , 3-Deoxy-7-Phosphoheptulonate Synthase/chemistry , 3-Deoxy-7-Phosphoheptulonate Synthase/metabolism , Allosteric Regulation , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Catalytic Domain , Chorismate Mutase/chemistry , Chorismate Mutase/genetics , Crystallography, X-Ray , Directed Molecular Evolution , Kinetics , Molecular Dynamics Simulation , Mutagenesis , Protein Structure, Quaternary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Alignment , Shikimic Acid/metabolism , Transition TemperatureABSTRACT
BACKGROUND: The induction of tolerance and protective immunity to microbes is significantly influenced by host- and microbiota-derived metabolites, such as histamine. OBJECTIVE: We sought to identify the molecular mechanisms for histamine-mediated modulation of pattern recognition receptor signaling. METHODS: Human monocyte-derived dendritic cells (MDDCs), myeloid dendritic cells, and plasmacytoid dendritic cells were examined. Cytokine secretion, gene expression, and transcription factor activation were measured after stimulation with microbial ligands and histamine. Histamine receptor 2 (H2R)-deficient mice, histamine receptors, and their signaling pathways were investigated. RESULTS: Histamine suppressed MDDC chemokine and proinflammatory cytokine secretion, nuclear factor κB and activator protein 1 activation, mitogen-activated protein kinase phosphorylation, and T(H)1 polarization of naive lymphocytes, whereas IL-10 secretion was enhanced in response to LPS and Pam3Cys. Histamine also suppressed LPS-induced myeloid dendritic cell TNF-α secretion and suppressed CpG-induced plasmacytoid dendritic cell IFN-α gene expression. H2R signaling through cyclic AMP and exchange protein directly activated by cyclic AMP was required for the histamine effect on LPS-induced MDDC responses. Lactobacillus rhamnosus, which secretes histamine, significantly suppressed Peyer patch IL-2, IL-4, IL-5, IL-12, TNF-α, and GM-CSF secretion in wild-type but not H2R-deficient animals. CONCLUSION: Both host- and microbiota-derived histamine significantly alter the innate immune response to microbes through H2R.
