scholarly journals RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production inSaccharomyces cerevisiae

2019 ◽  
Vol 116 (19) ◽  
pp. 9324-9332 ◽  
Author(s):  
Guokun Wang ◽  
Sara M. Björk ◽  
Mingtao Huang ◽  
Quanli Liu ◽  
Kate Campbell ◽  
...  
Keyword(s):  
Protein Secretion ◽  
High Throughput ◽  
Protein Modification ◽  
Protein Production ◽  
Enrichment Analysis ◽  
Cell Factory ◽  
Cellular Processes ◽  
Cellular Machinery ◽  
Specific Proteins

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeastSaccharomyces cerevisiaethat allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1,AAD4,ADE8, andSDH1), protein modification and degradation (VPS73,KTR2,CNL1, andSSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

2020 ◽  
Author(s):  
Valentin Romanov ◽  
Giulia Silvani ◽  
Huiyu Zhu ◽  
Charles D Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Adherent Cells ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Membrane Cytoskeleton ◽  
Change Temperature

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

scholarly journals A Proteomics Signature of Mild Hypospadias: A Pilot Study

2020 ◽  
Vol 8 ◽  
Author(s):  
Coriness Piñeyro-Ruiz ◽  
Horacio Serrano ◽  
Inmaculada Jorge ◽  
Eric Miranda-Valentin ◽  
Marcos R. Pérez-Brayfield ◽  
...  
Keyword(s):  
High Throughput ◽  
Energy Production ◽  
Isotope Labeling ◽  
Cellular Processes ◽  
Proteomics Approach ◽  
Liquid Chromatography Tandem Mass ◽  
Congenital Condition ◽  
Group A ◽  
Control Samples

Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients.Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples.Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias.Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

scholarly journals Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

2018 ◽  
Author(s):  
Eleni Mimitou ◽  
Anthony Cheng ◽  
Antonino Montalbano ◽  
Stephanie Hao ◽  
Marlon Stoeckius ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Surface Markers ◽  
Protein Levels ◽  
Cellular Processes ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Lymphoma Sample

ABSTRACTRapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma sample to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.

scholarly journals Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential

2021 ◽  
Author(s):  
Jolanda Neef ◽  
Jan Maarten van Dijl ◽  
Girbe Buist
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Protein Production ◽  
Cell Factory ◽  
Efficient Production ◽  
High Quality ◽  
Gram Positive ◽  
Secretion Pathway

Abstract Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium’s intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermentation broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current production bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped ‘non-classical’ protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the production of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein production in bulk.

Calcium/calmodulin-stimulated phosphorylation of photoreceptor proteins in Limulus

1989 ◽  
Vol 3 (2) ◽  
pp. 107-118 ◽  
Author(s):  
Eric M. Wiebe ◽  
Anne C. Wishart ◽  
Samuel C. Edwards ◽  
Barbara-Anne Battelle
Keyword(s):  
Protein Kinase ◽  
Dark Adaptation ◽  
Photoreceptor Cell ◽  
Cell Function ◽  
Photoreceptor Cells ◽  
Present Evidence ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Specific Proteins

AbstractCalcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse.In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.

scholarly journals Characterization of the Ubiquitin-Modified Proteome Regulated by Transient Forebrain Ischemia

2013 ◽  
Vol 34 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Masahiro Iwabuchi ◽  
Huaxin Sheng ◽  
JWill Thompson ◽  
Liangli Wang ◽  
Laura G Dubois ◽  
...  
Keyword(s):  
Protein Modification ◽  
Internal Standard ◽  
Forebrain Ischemia ◽  
Label Free ◽  
Cellular Processes ◽  
Damage Repair ◽  
Liquid Chromatography Tandem Mass ◽  
Posttranslational Protein Modification ◽  
Neuronal Functions

Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K- ε-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K- ε-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K- ε-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.

Combinatorial Fabrication and High-Throughput Characterization of Thin Film Metal Oxide Libraries for Solar water Splitting

2018 ◽  
Author(s):  
Alfred Ludwig ◽  
Mona Nowak ◽  
Swati Kumari ◽  
Helge S. Stein ◽  
Ramona Gutkowski ◽  
...  
Keyword(s):  
Thin Film ◽  
Metal Oxide ◽  
Water Splitting ◽  
High Throughput ◽  
Solar Water ◽  
Solar Water Splitting

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

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