scholarly journals Intelligent host engineering for metabolic flux optimisation in biotechnology

2021 ◽  
Vol 478 (20) ◽  
pp. 3685-3721
Author(s):  
Lachlan J. Munro ◽  
Douglas B. Kell
Keyword(s):  
Metabolic Networks ◽  
Metabolic Flux ◽  
Protein A ◽  
Search Space ◽  
Spare Capacity ◽  
Expression Levels ◽  
Regulatory Processes ◽  
A Cell ◽  
P Proteins ◽  
Combinatorial Sense

Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.

scholarly journals Division of labor in metabolic regulation by transcription, translation, acetylation and phosphorylation

2019 ◽  
Author(s):  
Sriram Chandrasekaran
Keyword(s):  
Metabolic Regulation ◽  
Metabolic Networks ◽  
Network Connectivity ◽  
Control Mechanisms ◽  
Post Translational Modifications ◽  
Regulatory Processes ◽  
Regulatory Architecture ◽  
A Cell ◽  
Metabolic Properties ◽  
Context Specific

AbstractThe metabolism of most organisms is controlled by a diverse cast of regulatory processes, including transcriptional regulation and post-translational modifications (PTMs). Yet how metabolic control is distributed between these regulatory processes is unknown. Here we present Comparative Analysis of Regulators of Metabolism (CAROM), an approach that compares regulators based on network connectivity, flux, and essentiality of their reaction targets. Using CAROM, we analyze transcriptome, proteome, acetylome and phospho-proteome dynamics during transition to stationary phase in E. coli and S. cerevisiae. CAROM uncovered that the targets of each regulatory process shared unique metabolic properties: growth-limiting reactions were regulated by acetylation, while isozymes and futile-cycles were preferentially regulated by phosphorylation. Reversibility, essentiality, and molecular-weight further distinguished reactions controlled through diverse mechanisms. While every enzyme can be potentially regulated by multiple mechanisms, analysis of context-specific datasets reveals a conserved partitioning of metabolic regulation based on reaction attributes.Author summaryThere are several ways to regulate an enzyme’s activity in a cell. Yet, the design principles that determine when an enzyme is regulated by transcription, translation or post-translational modifications are unknown. Each control mechanism, such as transcription, comprises several regulators that control a distinct set of targets. So far, it is unclear if similar partitioning of targets occurs at a higher level, between different control mechanisms. Here we systematically analyze patterns of metabolic regulation in model microbes. We find that five key parameters can distinguish the targets of each mechanism. These key parameters provide insights on specific roles played by each mechanism in determining overall metabolic activity. This approach may help define the basic regulatory architecture of metabolic networks.

Presence of antibody in virus-infected brain cells of SSPE ferrets

1983 ◽  
Vol 41 ◽  
pp. 804-805
Author(s):  
Hannah R. Brown ◽  
Tammy L. Donato ◽  
Halldor Thormar
Keyword(s):  
Measles Virus ◽  
Plasma Cells ◽  
Subacute Sclerosing Panencephalitis ◽  
Protein A ◽  
Neutralizing Antibody ◽  
Brain Cells ◽  
Antibody Titers ◽  
A Cell ◽  
The Central Nervous System ◽  
The Brain

Measles virus specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing disease of the central nervous system (CNS) in children. IgG/albumin ratios indicate that the antibodies are synthesized within the CNS. Using the ferret as an animal model to study the disease, we have been attempting to localize the Ig's in the brains of animals inoculated with a cell associated strain of SSPE. In an earlier report, preliminary results using Protein A conjugated to horseradish peroxidase (PrAPx) (Dynatech Diagnostics Inc., South Windham, ME.) to detect antibodies revealed the presence of immunoglobulin mainly in antibody-producing plasma cells in inflammatory lesions and not in infected brain cells.In the present experiment we studied the brain of an SSPE ferret with neutralizing antibody titers of 1:1024 in serum and 1:512 in CSF at time of sacrifice 7 months after i.c. inoculation with SSPE measles virus-infected cells. The animal was perfused with saline and portions of the brain and spinal cord were immersed in periodate-lysine-paraformaldehyde (P-L-P) fixative. The ferret was not perfused with fixative because parts of the brain were used for virus isolation.

scholarly journals Cell-Tak coating may cause mis-normalization of Seahorse metabolic flux data

2021 ◽  
Author(s):  
Michal Šíma ◽  
Stanislava Martínková ◽  
Anežka Kafková ◽  
Jan Pala ◽  
Jan Trnka
Keyword(s):  
Metabolic Flux ◽  
Cell Number ◽  
Cell Counting ◽  
Fluorescent Detection ◽  
Data Normalization ◽  
Tissue Samples ◽  
Flux Data ◽  
Research Areas ◽  
Healthy State ◽  
A Cell

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating may strongly affect the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

scholarly journals Multiscale 2D-SPR Biosensing for Cell Chips

2007 ◽  
Vol 19 (5) ◽  
pp. 519-523 ◽  
Author(s):  
Masayasu Suzuki ◽  
◽  
Toyohiro Ohshima ◽  
Shintaro Hane ◽  
Yasunori Iribe ◽  
...  
Keyword(s):  
Protein A ◽  
Cell Activity ◽  
Ccd Camera ◽  
Pdms Film ◽  
Sensor Film ◽  
Spr Imaging ◽  
Measurement Protocol ◽  
A Cell ◽  
Cooled Ccd ◽  
Imaging Sensor

Evaluating cell activity and functions in different-sized cell chambers requires multiscale sensing. We have been developing multiscale biosensing applied from 10 µm to 1 mm. We measured mouse IgG in micro wells using a high-resolution two-dimensional surface plasmon resonance (SPR) imaging affinity sensor. This sensor uses high refractive optics, a 1X to 7X microscopic lens, and a cooled CCD camera. The micro-well array was prepared with a PDMS film on gold sensor film. Protein A immobilized on sensor film was used for IgG recognition. SPR sensitivity was dramatically decreased with 10 and 8.5 µm microwells. To improve sensor sensitivity, we optimized the sensor’s measurement angle and exposure time, enabling mouse IgG to be detected in wells of 1 mm, 30 µm, and 10 µm using the same 2D-SPR imaging sensor and measurement protocol. These results show the feasibility of multiscale biosensing use in antibody production in a micro well or a cell chamber.

scholarly journals Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes

2021 ◽  
Author(s):  
Celine Deffrasnes ◽  
Meng-Xiao Luo ◽  
Linda Wiltzer ◽  
Cassandra T David ◽  
Kim G Lieu ◽  
...  
Keyword(s):  
Molecular Mapping ◽  
Protein A ◽  
Fine Tuning ◽  
Nuclear Trafficking ◽  
P Protein ◽  
Phenotypic Divergence ◽  
Reservoir Species ◽  
Pathogenic Viruses ◽  
P Proteins ◽  
Australian Bat Lyssavirus

Bats are reservoirs of many pathogenic viruses including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve P protein, a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in interferon/STAT1 antagonism and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct, and appear to involve modulatory effects on regulatory regions or structural impact, rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with ‘fine-tuning’ to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.

scholarly journals Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation

2015 ◽  
Vol 290 (47) ◽  
pp. 28299-28310 ◽  
Author(s):  
Shakur Mohibi ◽  
Shashank Srivastava ◽  
Jun Wang-France ◽  
Sameer Mirza ◽  
Xiangshan Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Protein A ◽  
Cell Cycle Regulator ◽  
A Cell

Pattern Analysis Meets Cell Biology

1999 ◽  
Vol 5 (S2) ◽  
pp. 510-511
Author(s):  
Robert F. Murphy ◽  
Michael V. Boland
Keyword(s):  
Cell Biology ◽  
Fluorescent Dyes ◽  
Protein A ◽  
Chinese Hamster ◽  
Subcellular Location ◽  
Computer Applications ◽  
Double Labeling ◽  
A Cell ◽  
Widespread Availability

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

scholarly journals A procedure for removing high titer antibodies by extracorporeal protein-A-sepharose adsorption in hemophilia: substitution therapy and surgery in a patient with hemophilia B and antibodies

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 38-44 ◽  
Author(s):  
IM Nilsson ◽  
S Jonsson ◽  
SB Sundqvist ◽  
A Ahlberg ◽  
SE Bergentz
Keyword(s):  
High Titer ◽  
Protein A ◽  
Factor Ix ◽  
Coagulation System ◽  
Substitution Therapy ◽  
Left Elbow ◽  
Tissue Antigens ◽  
A Cell ◽  
Total Immunoglobulin ◽  
Stimulation Of

Abstract A 37-yr-old hemophiliac with factor IX antibody in high titer required operation for a pseudotumor growing invasively into the bone in his left elbow. Exchange plasma-pheresis was not feasible, mainly because of possible stimulation of factor IX inhibitor. In addition, the patient had antibodies against tissue antigens. The factor IX antibodies were of IgG type and adsorbable to protein-A-agarose. The antibodies were removed by extracorporeal adsorption of the plasma (separated intermittently in a cell centrifuge) to sterile protein-A- Sepharose in columns, operated by a computer. The treated plasma was retransfused. In this manner, a total of 6000 ml of plasma was depleted of antibodies, and the antibody titer, as well as the total immunoglobulin content, decreased to one-fifth of the original values. The remaining antibodies were neutralized by infusion of factor IX concentrate. Conventional substitution therapy, in combination with immunosuppression, was then possible. The operation was performed without complications. No signs of hemolysis, complement activation, or activation of the coagulation system occurred. In principle, the same form of treatment can be used in patients with other forms of antibodies.

scholarly journals Endotoxin protein: a B-cell mitogen and polyclonal activator of C3H/HeJ lymphocytes.

1976 ◽  
Vol 144 (3) ◽  
pp. 821-827 ◽  
Author(s):  
B M Sultzer ◽  
G W Goodman
Keyword(s):  
B Cell ◽  
Lipid A ◽  
Protein A ◽  
Cell Wall Protein ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Lipopolysaccharide Endotoxin ◽  
Polyclonal Activator ◽  
A Cell ◽  
Cell Mitogen

A cell wall protein that is ordinarily complexed to the lipopolysaccharide endotoxin in gram-negative bacteria has been separated by the use of aqueous phenol. The protein is active as a B-cell mitogen and polyclonal activator of murine lymphocytes including the C3H/HeJ strain which is a nonresponder to lipoplysaccharide or lipid A.

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