Inducible Protein

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.

Vaccine candidate for newly emerged H7N9 influenza virus

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.

A breakthrough in cancer treatment: GLUT1 crystal structure

GLUT1 Structure
Overall structure of the human glucose transporter GLUT1

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

GLUT1 Inhibitor
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

The Difficult Of Eukaryotic Genes Expression By Bacteria

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.

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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.

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Algae virus can jump to mammalian cells

ATCV-1 viral particles
This optical image shows a cell (in blue) with the ATCV-1 viral particles.
Credit: University Communications/University of Nebraska-Lincoln

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.

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Bacterial protein can help convert stem cells into neurons

neuronsAs 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.

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Alzheimer’s Disease breakthrough: Humam Gamma secretase structure

γ-secretase complex crystal structure
An overall density map for the entire human Gamma secretase structure.

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.




IPTG Induction Protocol

IPTG induction in bacteria can be performed using one of two basic methods. Fast induction does not work for all proteins and can give you suboptimal yields. Slow induction can enhance the solubility of some proteins. The method that is best for you will depend on your particular protein and the application. If you want optimal solubility both should be tested before scaling up. This IPTG induction protocol is generalized and will vary based on a variety of factors such as the bacterial strain, recombinant protein, and parent plasmid.

Fast IPTG induction protocol

  1. From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 30C (or 37C) in 1-2ml LB+AMP (or other selection) in a 15ml snap cap tube on a rotator or shaker.
  2. Dilute 1:50 (1:100 if 37C O/N) in 2ml LB+AMP and grow 3-4 hours at 37 C in 15ml snap cap tube in a rotator.
  3. Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use.
  4. After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. Spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed. THIS IS THE UNINDUCED CONTROL.
  5. Add prewarmed 1ml LB+AMP+1mM IPTG to 15ml snap cap tube and return to 37 C for 3-4 hours. This will get the final volume back to 2ml and the final concentration of IPTG to 0.5mM.
  6. After 3-4hrs transfer 1ml from induced sample to labeled 1.5ml tubes and spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed. THIS IS THE INDUCED SAMPLE.
  7. Sample preparation for SDS-PAGE: Add 100ul of 1X loading buffer (see solutions below) with 1% BME to uninduced and induced samples. Vortex for 10sec to 1min or until there are no clumps of bacteria. Boil 3-5min, spin at max, 30sec, RT, and load 5-25ul (usually 10ul) depending on gel (amount of protein, size of pellet, Western, etc.).

Slow IPTG induction protocol

For slow IPTG induction protocol of protein follow fast IPTG induction protocol with the following changes:

  • 6. Add 20 C 1ml LB+AMP+1mM IPTG to 15ml snap cap tube and incubate rotating or shaking at 20 C for 12-16 hours.  This will get the final volume back to 2ml and the final concentration of IPTG to 0.5mM.
  • 7. After 12-16hrs transfer 1ml from induced sample to labeled 1.5ml tubes and spin at max, 30sec, RT, and remove supe.  Freeze pellet at -20 until needed. THIS IS THE INDUCED SAMPLE.

NOTES for induction:

*Induction times vary from 2-5hrs.

*IPTG can be varied from 0.1-1.0M.

*If you boil your sample too long they will become viscous from total release of cellular DNA. You can still use them if you can find an area of low viscosity, however, its usually better just to repeat the experiment.

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Use in laboratory

IPTG is an effective inducer of protein expression in the concentration range of 100 μM to 1.0 mM. Concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used. If lacIq, a mutant that over-produces the lac repressor, is present, then a higher concentration of IPTG may be necessary.

In blue-white screen, IPTG is used together with X-gal. Blue-white screen allows colonies that have been transformed with the recombinant plasmid rather than a non-recombinant one to be identified in cloning experiments.

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IPTG Induction Protein Expression

Isopropyl β-D-1-thiogalactopyranoside (IPTG, also known as lad-y) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce recombinant protein expression where the gene is under the control of the lac operator.
Like allolactose, IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. But unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer. The concentration of IPTG therefore remains constant and the expression of lac p/o-controlled genes would not be inhibited during the experiment.

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The lac operon is one of the most commonly used systems for creating recombinant proteins in E. coli that can then be purified and studied in other experiments. Usually your gene of interest is inserted into a commercial vector (pET vectors are common) that contains:

  1. A gene coding for antibiotic resistance
  2. The lacI gene from the lac operon that codes for the lac repressor (LacI)
  3. Your gene of interest inserted just after the T7 promoter DNA sequence, the lac operator DNA sequence, and the ribosome binding site (at the start of the future mRNA transcript).

IPTG Induction

The lac repressor protein (LacI) evolved to sense the presence of lactose (a combined galactose-glucose disaccharide). Both the host chromosome and the insert have copies of the lac repressor gene to ensure that there is always enough LacI protein to titrate all DNA operator sites. In the absence of lactose, the lac repressor binds to the operator sequence on DNA and bends the DNA by 40 degrees. This blocks access of T7 RNA polymerase to the promoter site and thus prevents leaky transcription of your gene before induction.

When lactose binds to LacI it induces a conformational change in the protein structure that renders it incapable of binding to the operator DNA sequence. IPTG is a structural mimic of lactose (it resembles the galactose sugar) that also binds to the lac repressor and induces a similar conformational change that greatly reduces its affinity for DNA. Unlike lactose, IPTG is not part of any metabolic pathways and so will not be broken down or used by the cell. This ensures that the concentration of IPTG added remains constant, making it a more useful inducer of the lac operon than lactose itself.

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Protein Folding and Protein Structure Prediction Problem

Protein Structure Introduction And Protein Fold problem

protein 3D structure prediction
Protein Structure Prediction (PSP) aims to predict the 3D structure of a protein based on its primary sequence.(picture from: )

Proteins accomplish their task by three-dimensional tertiary and quaternary interactions between various substrates such as DNA and RNA, and other proteins. Thus knowing the structure of a protein is a prerequisite to gain a thorough understanding of the protein’s function.

Knowing the structure of a protein sequence enables us to probe the function of the protein, understand substrate and ligand binding, devise intelligent mutagenesis and biochemical protein engineering experiments that improve specificity and stability, perform rational drug design, and design novel proteins. Understanding structure has potential applications in the various genome projects being undertaken, such as mapping the functions of proteins in metabolic pathways for whole genomes and deducing evolutionary relationships. The protein folding problem is therefore one of the most fundamental unsolved problems in computational molecular biology today.

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The Difficult Of Predicting Protein Fold

  1. Force fields are “bad” – but it is getting better. “Systematic Validation of Protein Force Fields against Experimental Data” is a good paper to read.
  2. It takes a very long time to generate the trajectories (the paths a protein undertakes). Numerous approaches have been suggested, including sampling hundreds of thousands of trajectories at once , or simulating a single extremely long trajectory. They have different uses. By and large, the biggest bottle neck in MD simulation is the evaluation  of the pair-wise electrostatic interactions. Naive implementations require O(N^2) time, particle mesh based methods can accomplish this in O(N log N) time. All the other steps in MD.
  3. Analysis and interpretation of data. This is a really finnicky one. There are many many different ways to dissect and analyze a trajectory. A definition of a “minima”, or a “convex” shape, per your terminology, does not easily generalize to a higher dimensional space.

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