Most high yield E. coli protein production systems are with the target gene under an inducible promoter. The strain is first cultivated under healthy growth conditions in order to build up the cell mass. The culture is then subjected to an induction, whether it’s a physical event such as a temperature shift or a chemical inducer such as a sugar. The target protein starts to express in large amount. The cells continue to grow, but in a somewhat slower rate. Usually in a short period of time thereafter, the expression reaches the maximum level. Cells will then be harvested and processed when the protein production yield no longer increases or cells cease to grow any further.
For our selection of strategy, the over-expressed protein should be a robust one that is easy to quantify, not necessarily a high value target. We choose E. coli lacZ encoded beta-galactosidase. It’s a widely used reporter for gene transcription and recombinant protein translation, without known toxic effect on cellular functions. It’s an enzyme that can be easily and accurately assayed, which means we may reliably capture even a small percentage of increase of beta-galactosidase expression. [A small increase, such as a mere 5%, may mean little in a laboratory setting but not so in manufacturing.
As explained above, we will create conditions to decrease cell growth around the time of induction. There are two key parameters for optimization. (1) At what time, relative to the induction point, should the induced slow growth occur (before, at the same time, or even after)? Most likely, a preemptive one should be the best. Then the question is how early the slow growth should be induced. (2) To what degree, relative to the normal growth rate before and after the beta-galactosidase over-expression, should the cell growth rate decrease? We will optimize both of these parameters in each of the following maneuvers.
iBio, Inc. has produced a vaccine candidate for the newly emerged H7N9 influenza virus by an independent third party laboratory using the iBioLaunch platform. This validation milestone was achieved in 21 days as measured from initial antigen sequence information to purification of recombinant protein.
The iBioLaunch platform eliminates the need to culture cells under sterile conditions, removes uncertainty about yield consistency for large volumes of production, and, subject to regulatory approval, could deliver vaccine doses for emergency use against pandemic and bioterrorism threats in weeks rather than the months necessary with the use of engineered or attenuated virus strains. The iBioLaunch platform has been used previously to produce vaccine-quality antigens associated with a broad range of influenza strains including H7N7, H5N1, H3N2, H1N1, and various strains of influenza B.
These vaccine applications of the iBioLaunch platform are of particular interest to governments and state corporations that seek autonomy in the manufacture of critical biopharmaceuticals to protect their own populations.
The H7N9 influenza virus has killed 36 of 131 people reported to have been infected in China. The emergence of new influenza and other virus strains is a constant public health risk that is only partially addressed with commonly used technologies. Because of the slowness of conventional approaches to vaccine development and production, the only effective control measures currently employed in many circumstances involve patient isolation and supportive care.
iBio develops and offers product applications of its iBioLaunch and iBioModulator platforms, providing collaborators full support for turn-key implementation of its technology for both proprietary and biosimilar products.
GLUT1 crystal structure
please go to Read more in nature
Introduction of GLUT1 transporter
Glucose transporter 1 (or GLUT1) with highly conservatism, facilitates the transport of glucose across the plasma membranes of mammalian cells. read more in wikipedia
WZB117 – A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo.
Fasentin – a small molecule inhibitor of the intracellular domain of GLUT1 preventing glucose uptake.
GLUT1 Deficiency syndrome
GLUT1 deficiency syndrome is a disorder affecting the nervous system that can have a variety of neurological signs and symptoms. GLUT1 deficiency syndrome is a rare disorder. Approximately 500 cases have been reported worldwide since the disorder was first identified in 1991. read more
When bacteria are used to produce a eukaryotic protein, it is desirable to design the system so as to produce as large an amount of the protein as possible.
There are several such systems for overproducing foreign proteins in E. coli. For instance, One system uses phage T7 RNA polymerase operating on a T7 promoter. Late in infection, phage T7 synthesizes enormous amounts of gene products from several sites termed late promoters. Because host chromosomal genes are not synthesized at this point, these products are the only proteins synthesized. The T7 RNA polymerase then transcribes this gene at high levels, resulting in high production of the factor VIII protein in the bacteria.
More details from: http://www.ncbi.nlm.nih.gov/books/NBK21359/
Prokaryotic cells (bacteria, especially E. coli) are normally the preferred host for the expression of foreign proteins because they offer: Inexpensive carbon source requirements for growth, Rapid biomass accumulation, Amenability to high-cell density fermentation, and Simple process scale up.
However, a lack of post-translational machinery and production of inactive protein due to the formation of inclusion bodies. One disadvantage of using an organism such as E. coli for expression of eukaryotic genes is that it is a prokaryote, and therefore lacks the membrane-bound nucleus (and other organelles) found in eukaryotic cells. This means that certain eukaryotic genes may not function in E. coli as they would in their normal environment, which can hamper their isolation by selection mechanisms that depend on gene expression.
More details from: https://www.quora.com/What-makes-it-difficult-for-a-eukaryotic-protein-to-be-synthesized-by-bacteria
New research led by the University of Nebraska-Lincoln has provided the first direct evidence that an algae-infecting virus can invade and potentially replicate within some mammalian cells.
Known as Acanthocystis turfacea chlorella virus 1, or ATCV-1, the pathogen is among a class of chloroviruses long believed to take up residence only in green algae.The new study, introduced ATCV-1 to macrophage cells that serve critical functions in the immune responses of mice, humans and other mammals. By tagging the virus with fluorescent dye and assembling three-dimensional images of mouse cells, the authors determined that ATCV-1 successfully infiltrated them.
Though a few studies have documented viruses jumping from one biological kingdom to another, chloroviruses were previously thought to have a limited “host range” that stopped well short of the animal kingdom.
The macrophage cells underwent multiple changes characteristic of those breached by a virus, Dunigan said. These changes eventually included a form of programmed death that virologists consider an innate “scorched earth” defense against the spread of viruses, which require living cells to survive and replicate.
Before dying, the cells exhibited multiple signs of stress that tentatively support links to mild cognitive impairments. The new study measured a post-viral rise in interleukin 6, a cellular protein that previous research has linked with diminished spatial learning and certain neurological diseases.
The new study’s authors are continuing their collaboration with Johns Hopkins in the hope of ultimately confirming whether and how the virus contributes to any cognitive deficits suggested by the initial studies.
“It is still unclear whether the factors induced by the cell-based virus challenge could also be induced in the whole animal, and whether the induced factors cause cognitive impairments in the animal or the human,” said co-author Tom Petro, professor of microbiology and immunology at the University of Nebraska Medical Center.
More details from: https://www.sciencedaily.com/releases/2015/10/151021185110.htm
As the recipe book for turning stem cells into other types of cells keeps growing larger, the search for the perfect, therapeutically relevant blend of differentiation factors is revealing some interesting biology. A new study, for example, found that a protein in E. coli bacterial combined with small molecules can act synergistically to push pluripotent cells into functional neurons.
A study published November 19 in Chemistry & Biology, for example, found that a protein in E. coli bacteria combined with small molecules can act synergistically to push pluripotent cells into functional neurons. The differentiation of pluripotent stem cells can be conceived as two simple steps: first, a stem cell decides to no longer be a stem cell and begins to differentiate; second, the cell decides what kind of cell it wants to be. In protocol to induce neuron differentiation, the bacterial protein Skp acts in the first step by binding to Sox2 and inhibiting its function. The small chemicals neurodazine (Nz) and neurodazole (Nzl) then act in the second step by telling the stem cell to become a neuron. By influencing both steps, more functional neurons can be produced per batch of stem cells and at a faster rate if using either protein or small molecules alone.
One weakness of the protocol is that there are safety concerns around using bacterial proteins such as Skp in a therapeutic setting. However, using this protein is advantageous compared to introducing genetic elements because protein cannot cause any genetic alteration or instability, which are the major concerns of using virus-mediated gene delivery to the stem cells.
More details from: https://www.sciencedaily.com/releases/2015/11/151119133237.htm
Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian expression system. Transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. Transient gene expression in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells.
Transient transfection introduction from: https://www.researchgate.net/publication/
For some applications of transient transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution.
Transient transfection introduction from: https://en.wikipedia.org/wiki/Transfection#Stable_and_transient_transfection
The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer’s disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 Å resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex.
Recombinant protein purification is a widely used method for purifying proteins of interest based on well-developed recombinant DNA and protein expression technologies. Theoretically, all proteins can be purified using this method regardless of the solubility of the proteins produced in expression host cells.
When a recombinant protein is fused to a peptide or protein tag, such as polyhistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP), or Strep-tag II, the properties of the tag can be exploited for purification purposes. Affinity chromatography methods have been developed for each of the commonly used tags, and there is a good chance of a successful purification of a tagged protein in a single step.
Protein fusion tags are indispensible tools used to improve recombinant protein purification yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts.
Useful applications of affinity-tag purification schemes include:
The two most commonly used tags are the polyhistidine tag generally consisting of six 10 histidine residues and GST.
Proteins tagged with histidine bind strongly with metal ions, such as Ni2+, Co2+, Cu2+, and Zn2+. They and are purified by binding to a metal ion immobilized on a support resin by the IMAC method (immobilized metal ion affinity chromatography). Either end of the recombinant protein can be tagged with histidine. Optimal placement is empirically determined and can vary from protein to protein.
For GST tags, the purification principle is based on the binding of GST to gluthathione immobilized on the support resin. After sample impurities are washed from the resin, the bound GST-tagged protein is eluted by reduced glutathione. The GST protein has a molecular weight of 26 kD and is most often fused to the target protein at the N-terminus, though it can work well with C-terminal fusions. The GST protein is a dimer in solution and, thus, the fusion protein dimerizes as well. GST tags can be used to increase solubility of the recombinant protein.
Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins.
Soluble protein definition from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544838/
The soluble protein test procedure is based on attempting to dissolve chemicals in various solvents with a increasingly rigorous mechanical techniques. The solvents to be used, in the order of preference, are cell culture media, DMSO, and ethanol. Solubility shall be determined in a step-wise procedure that involves attempting to dissolve a test chemical in the solvents (in the order of preference) at relatively high concentrations using the sequence of mechanical procedures. If the chemical does not dissolve, the volume of solvent is increased so as to decrease the concentration by a factor of 10, and then the sequence of mechanical procedures are repeated in an attempt to solubilize the chemical at the lower concentrations.
Soluble protein test introduction from: https://ntp.niehs.nih.gov/
1. Take the remaining tube of induced cells and resuspend in 50 uL of B-PER containing protease inhibitors (PMSF or Complete).
2. Incubate at room temperature for 10 mins.
3. Spin down in a microcentrifuge at maximum speed for 10 min at 4 ºC.
4. Carefully transfer all of the supernatant into a new microfuge tube. Add 50 uL of 2x SDS-PAGE buffer. This is the soluble fraction.
5. Resuspend the pellet in 100 uL of 1x SDS-PAGE buffer. This is the insoluble fraction.
6. Boil the samples for 10 min, then cool down to room temperature.
7. Centrifuge for 5 mins at maximum speed at room temperature.
8. Analyze 15 uL of each sample using SDS-PAGE, with western blotting if necessary.
This soluble protein test protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.
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