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Are the terms gene expression assays and protein expression assays used interchangeably in molecular biology? Or is it expected of you to differentiate between the two terms.
For example, if I replace the DNA sequence of a gene of interest with the GFP DNA; and then subsequently quantify the fluorescence levels, am I measuring the gene expression of the gene of interest OR the protein expression?
Gene expression is the production of a functional gene product from a gene. So protein expression is a functional read-out of gene expression for gene loci that encode proteins.
However, gene expression is frequently, and erroneously, used to refer to measures of mRNA levels. The production of mRNA is a prerequisite step for the production of protein from a gene and it provides information about the transcriptional activity of a gene loci.
Translation of mRNA is a highly regulated process and global assays highlight that mRNA transcript levels explain less than half of the variation in protein concentrations.
For example, if I replace the DNA sequence of a gene of interest with the GFP DNA; and then subsequently quantify the fluorescence levels, am I measuring the gene expression of the gene of interest OR the protein expression?
You are measuring both gene expression and protein expression from the promoter + GFP gene loci. If you are interested in transcription from the promoter then measuring transcript would provide a more direct measure.
Gene expression and protein expression are absolutely not the same thing. While counts of mRNA levels for a particular gene may give some indication of how much protein is present, many times it does not. Similarly, quantitating protein levels does not necessarily mean the relative levels of mRNA will be similar. There are many points for regulation between RNA polymerase rolling along the gene and creating the pre-mRNA and the protein coming out of the ribosome and being folded and transported to its correct place in the cell. mRNA can be silenced, degraded, or prevented from being translated by a number of mechanisms. Proteins themselves are subject to extensive regulation, with half-lives that can vary from seconds to weeks. Of course, genes are regulated in many different ways as well, from repressor proteins to methylated DNA to chemically-modified histones, and the number of polymerase complexes on the DNA and their relative transcription speed are also under regulatory control.
If I replace the DNA sequence of a gene of interest with the GFP DNA; and then subsequently quantify the fluorescence levels, am I measuring the gene expression of the gene of interest OR the protein expression?
You are quantifying the protein levels of GFP. mRNAs can be subject to sequence-specific regulation (such as siRNA), so you can't just say absolutely that GFP levels are equal to your gene product of interest. Many times they will be proportional, but some times they won't, so you'll need to do a number of other experiments to verify your results.
Gene expression workflow.
Researchers may perform gene expression analysis at any one of several different levels at which gene expression is regulated: transcriptional, post-transcriptional, translational, and post-translational
Transcription, the process of creating a complementary RNA copy of a DNA sequence, can be regulated in a variety of ways. Transcriptional regulation processes are the most commonly studied and manipulated in typical gene expression analysis experiments.
The binding of regulatory proteins to DNA binding sites is the most direct method by which transcription is naturally modulated. Alternatively, regulatory processes can also interact with the transcriptional machinery of a cell. More recently, the influence of epigenetic regulation, such as the effect of variable DNA methylation on gene expression, has been uncovered as a powerful tool for gene expression profiling. Varying degrees of methylation are known to affect chromatin folding and strongly affect accessibility of genes to active transcription.
Following transcription, eukaryotic RNA is typically spliced to remove noncoding intron sequences and capped with a poly(A) tail. At this post-transcriptional level, RNA stability has a significant effect on functional gene expression, that is, the production of functional protein. Small interfering RNA (siRNA) consists of double-stranded nucleic acid molecules that are participants in the RNA interference pathway, in which the expression of specific genes is modulated (typically by decreasing activity). Precisely how this modulation is accomplished is not yet fully understood. A growing field of gene expression analysis is in the area of microRNAs (miRNAs), short RNA molecules that also act as eukaryotic post-transcriptional regulators and gene silencing agents.
What is a Gene?
Gregor Mendel was the first person to describe the existence of genes and their inheritance patterns. He explained the inheritance of traits in terms of inherited characteristics and did not use the term ‘gene’. The term ‘Gene’ is lately evolved with the development of Genetics. Gene is a segment of DNA, which contains instructions to form proteins. Each gene has a specific sequence of base pairs, which determines the structure and function of a specific protein. Genes are the blueprints of all the traits in the body. They determine most of the characteristic features of organisms and are able to pass these characteristic features to next generations the process called heredity. These characteristic features are known as traits, some of which are visible and some are not.
Bioluminescent Firefly Luciferase Assays
Firefly luciferase is a widely used bioluminescent reporter for studying gene regulation and function. It is a very sensitive genetic reporter due to the absence of endogenous luciferase activity in mammalian cells or tissues. Firefly luciferase is a 62,000 Dalton protein, which is active as a monomer and does not require subsequent processing for its activity. The enzyme catalyzes ATP-dependent D-luciferin oxidation to oxyluciferin, producing light emission centered at 560 nm. Light emitted from the reaction is directly proportional to the number of luciferase enzyme molecules.
Figure 1. Luciferase assay principle. Bioluminescent reaction catalyzed by firefly luciferase produces light.
What is the Difference Between Bioluminescence and Fluorescence?
Bioluminescence is a chemical process in which an enzyme breaks down a substrate (such as Luciferase and D-luciferin) and one of the by-products of this reaction is light. Bioluminescence naturally occurs in nature in various algae, bacteria, fungi and some aquatic animals such as jellyfish. Fluorescence is a physical process by which light excites electrons in the fluorophore to a higher energy state, and when that electron falls back down to its ground state it emits a photon.
How do Luciferase Assays Work?
Firefly luciferase assays (SCT151, SCT154, LUC1, LUCASSY-RO) are designed for simple and efficient quantitation of firefly luciferase reporter enzyme activity from cultured cells with high sensitivity and linearity. This is a flash-type luminescence assay that requires signal to be measured immediately after adding working solution to samples. The luminescence signal decays over the course of about 10 minutes of reaction time, although signal half-life may vary depending on luciferase expression levels.
The light production resulting from the luciferase reaction leads to formation of suicidal adenyl-oxyluciferin at the enzyme surface. It results in very short half-life of the light emission with a flash-type kinetics. The Firefly Luciferase HTS Assay (SCT150) is designed with a proprietary mixture of substances that modify the enzymatic reaction to produce a long-lasting signal (steady-glow) by preventing the formation of adenyl-oxyluciferin at the enzyme surface. It is a homogeneous high sensitivity firefly luciferase reporter gene assay kit for the quantification of firefly luciferase expression in mammalian cells with signal half-life of about 3 hours (Figure 2). Glow-type luciferase assays have lower luminescence signal compared to flash-type assays. The sensitivity and limit of detection of the assay will depend on luciferase expression levels in your experimental system as well as luminometer sensitivity.
Figure 2. Titration of recombinant firefly luciferase in the firefly luciferase assay. A) Recombinant luciferase was serially diluted in 1X Firefly Lysis Buffer with 1 Mg/mL BSA and measured in the assay. B) The Firefly Luciferase HTS Assay is a steady-glow high sensitivity firefly luciferase reporter gene assay kit for the quantification of firefly luciferase expression in mammalian cells with signal half-life of about 3 hours.
What is the Difference Between Firefly and Renilla Luciferase?
Firefly luciferase assays uses luciferin in the presence of oxygen, ATP and magnesium to produce light (Green/Yellow, 550-70 nM), while Renilla luciferase assays (SCT153) requires only coelenterazine and oxygen to produce light (Blue, 480 nM). Renilla luciferase has been used as a reporter gene for studying gene regulation and function in vitro and in vivo. It commonly is used in multiplex transcriptional reporter assays or as a normalizing transfection control for firefly luciferase assays. The enzyme does not require post-translational modification for its activity and may function as a genetic reporter immediately following translation. Coelenterazine, substrate for Renilla luciferase, also emits light from enzyme-independent oxidation, a process known as autoluminescence. The autoluminescence is enhanced by superoxide anion and peroxynitrite in cells and tissues.
Figure 3. Firefly vs. Renilla Luciferase. Bioluminescent reactions catalyzed by firefly luciferase and Renilla luciferase.
What are the Advantages of Dual Luciferase Assays?
The Firefly/Renilla Dual Luciferase Assay (SCT152) allows measurement of both Firefly and Renilla luciferase activity in the same sample with high sensitivity and linearity. Firefly luciferase activity is measured first, then Renilla Luciferase Assay Buffer 2.0 is added to simultaneously quench firefly luciferase activity and measure Renilla luciferase activity. Renilla Luciferase Assay Buffer 2.0 quenches the firefly luciferase activity to the level of un-transfected cells, allowing sequential measurement of firefly and Renilla luciferase activity in the same sample. This is a flash-type assay that requires luminescence to be measured immediately after adding the detection reagents to the luciferase sample. Firefly signal decays over the course of about 12 minutes, while Renilla signal decays over the course of about 2 minutes, although this may vary depending on enzyme levels.
Figure 4. Dual luciferase assay overview. Example of Firefly & Renilla Luciferase detection using lysates from untransfected HeLa cells or cells transfected with either firefly luciferase alone (Firefly Only) or co-transfected with firefly and Renilla luciferases (Firefly + Renilla). In cells transfected with firefly only, the Renilla signal is the residual firefly luminescence after adding Renilla working solution to the reaction.
What are the Applications of Luciferase Assays?
Genetic reporters are used as indicators to study cellular events coupled to gene expression. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector (which carries a Luciferase gene) that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic luciferase activity. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).
For example, Sigma and SwitchGear Genomics™ have teamed up to exclusively offer a genome-wide collection of over 10,000 3′UTR regions in our optimized lentivirus luciferase reporter vector system. When conducting experiments using MISSION ® 3′UTR Lenti GoClones, including the proper controls permits accurate interpretation of the experimental gene expression results.
Cell Viability via ATP Detection
Because ATP is an indicator of metabolically active cells, the number of viable cells can be assessed based on the amount of ATP available. The ATP Cell Viability Luciferase Assay (SCT149, 11699709001) offers a highly sensitive homogenous assay for quantifying ATP in cell cultures. This kit takes advantage of Firefly luciferase’s use of ATP to oxidize D-Luciferin and the resulting production of light in to assess the amount of ATP available in cell cultures. The sensitive assay procedure involves a single addition of ATP detection cocktail directly to cells cultured in a serum-supplemented medium. No cell washing, medium removal and multiple pipetting are required. The kit can be used to detect as little as a single cell or 0.01 picomoles of ATP. The signal produced is linear within 6 orders of magnitude. By relating the amount of ATP to the number of viable cells, the assay has wide applications, ranging from the determination of viable cell numbers to cell proliferation to cell cytotoxicity.
Figure 5. ATP cell viability assay. Firefly luciferase’s use of ATP to oxidize D-Luciferin and the resulting production of light in order to assess the amount of ATP available that correlates to cell number and viability.
In vivo Imaging
Bioluminescence imaging (BLI) has become a popular technique for optical tracking of cells in small laboratory animals such as mice. Along with the low background autofluorescence and cellular toxicities, BLI can enable simultaneous visualization of monitoring for the expression of two divergent luciferase proteins by use of their specific substrates. The imaging dual luciferase gene (red codon optimized firefly luciferase and a green click beetle luciferase) activities in the same animals could reduce variations from individual differences of the experimental animals.
Expression Analysis and Binding Assays in the Chemosensory Protein Gene Family Indicate Multiple Roles in Helicoverpa armigera
Chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals to receptors on sensory neurons. We identified and cloned 24 CSP genes to better understand the physiological function of CSPs in Helicoverpa armigera. Quantitative real-time polymerase chain reaction assays indicate that CSP genes are ubiquitously expressed in adult H. armigera tissues. Broad expression patterns in adult tissues suggest that CSPs are involved in a diverse range of cellular processes, including chemosensation as well as other functions not related to chemosensation. The H. armigera CSPs that were highly transcribed in sensory organs or pheromone glands (HarmCSPs 6, 9, 18, 19), were recombinantly expressed in bacteria to explore their function. Fluorescent competitive binding assays were used to measure the binding affinities of these CSPs against 85 plant volatiles and 4 pheromone components. HarmCSP6 displays high binding affinity for pheromone components, whereas the other three proteins do not show affinities for any of the compounds tested. HarmCSP6 is expressed in numerous cells located in or close to long sensilla trichodea on the antennae of both males and females. These results suggest that HarmCSP6 may be involved in transporting female sex pheromones in H. armigera.
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Comparatively Speaking: Gene vs. Protein Expression Studies
What is the difference between gene and protein expression studies to evaluate the efficacy cosmetic products? In this edition of our "Comparatively Speaking" column, Tony O&rsquoLenick asked Nava Dayan, Ph.D., for her thoughts on the topic the following is her response:
When scientists worldwide began to collaborate in the mapping of the entire human genome, they were under impression that we have more than the approximately 25,000 genes that we actually carry. Perhaps it was human arrogance to think that we are somehow more biologically superior to other species than we truly are. In the process of unraveling our complete genomic profile, we came to the realization that we attributed to genes more control than they actually have.
The &ldquocentral dogma of molecular biology,&rdquo coined by Francis Crick in 1958&mdashin essence, that one DNA is translated to one RNA, and one RNA is translated to one protein that exhibits one function&mdashhas been proven inaccurate. In reality, there is more to the epigenome (where &ldquoepi&rdquo means beyond, above) in controlling biological functioning than to the genome.
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In our industry, companies conduct gene expression studies and present those to imply certainty in the effect of their raw materials and formulations on the skin&rsquos biology. It is important to understand the limitations of such data set and to consider them in the correct context. The technical term &ldquogene expression&rdquo merely means that a DNA sequence is translated in the cell nucleus to messenger RNA (mRNA). When a specific gene is &ldquoup regulated,&rdquo it means that the amount of mRNA generated from that sequence is higher in quantity than normal when it is &ldquodown regulated,&rdquo it means that it is lower when compared to a normal state.
The nomenclature for genes and protein is similar, and is composed from a combination of letters and numbers for example, interleukin 1 alpha is abbreviated to IL-1a, and tumor necrosis factor is abbreviated to TNF-a. The translation of DNA to mRNA is a step into a production of peptides and proteins&mdashmRNA may or may not be translated to a protein. The peptides and proteins are small or larger sequences of amino acids, respectively, and are the &ldquoworking horses&rdquo of our body. They act as cytokines to deliver messages, as enzymes to facilitate energy efficient biochemical cascades and as receptors to communicate between different cellular compartments and between the cell and its environment.
With today&rsquos technological tools, we can quantify and qualify genes and proteins expression. However, it is a key to understand that if a gene is being expressed it does not completely equate activity since it further needs to be translated to a protein. Moreover, even if the protein is being created, it needs an adequate environment to impart that appropriate activity. The mere 3-D structure of a protein that is dictated by its environment will affect its &ldquopost-translational modifications&rdquo that is determined by the environment it is in.
Therefore, when conducting an in vitro study for efficacy screening, methods used should be considered and evaluated knowing its advantages and limitations. With today&rsquos technology and tools, a cost-effective and meaningful study can be tailored to maximize output.
- During chemical biotin-labeling the protein can become inactivated due to random biotinylation of the protein surface by the attachment of biotin to protein catalytic or binding domains. With Avi-Tag, virtually any protein can be easily and efficiently biotinylated in vivo or in vitro using the single, unique AviTag site.
- Biotinylation using Avi-Tag is performed enzymatically resulting in gentle reaction conditions and highly specific labeling.
- Biotin-AviTag has 15 amino acids, which is a fifth of the bulk of alternative biotinylation tag sequences that are over 85 amino acid residues long an important consideration if steric conflicts are to be minimized.
Figure1. OmicsLink ORF cDNA expression clones with N- and C-terminus AviTag in various mammalian vector systems.
How it works
Reporter gene assays
A reporter gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection of the reporter protein or its enzymatic activity is measured. The effect of promoters or enhancer regions on gene expression can be determined by detection of the reporter in a specific assay, which ideally would have low background signal, high sensitivity and of course be quick, accurate and safe. In the case of a luciferase assay, photon emission is measured resulting from the catalysis of a chemical reaction requiring luciferin, ATP and oxygen as substrates. Production of photons by this bioluminescent reporter occurs slower than fluorescent-based methods, such as excitation of Green Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity laser to rapidly excite GFP. As a result of the different mechanisms to produce photons, chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage of lower background levels and improved signal sensitivity since photons are simply measured – they are not required to initiate the reaction. Table 1 compares some general advantages and disadvantages of luminescence versus fluorescence .
Table 1: Properties of Luminescence versus Fluorescence
|Source of Emitted Photons||Chemical reaction||High-energy photons|
|Kinetics of Photon Generation||Slower||Faster|
|Post-translational Modification||Not Required||Required|
High-Titer Recombinant Protein Production
Henry Chiou Ph.D. Associate Director Thermo Fisher Scientific
Jonathan F. Zmuda Ph.D. Director of R&D Thermo Fisher Scientific
Maya Yovcheva R&D Scientist and R&D Lead Thermo Fisher Scientific
Kenneth Thompson Ph.D. R&D Scientist Thermo Fisher Scientific
Sara Barnes R&D Scientist Thermo Fisher Scientific
Katalyn Irvin R&D Scientist Thermo Fisher Scientific
Melissa Cross R&D Scientist Thermo Fisher Scientific
Natasha Lucki Ph.D. Product Manager Thermo Fisher Scientific
Employing the ExpiSf™ Chemically Defined Sf9 Insect Cell Expression System
The baculovirus expression vector system (BEVS) provides a versatile platform for the expression of individual recombinant proteins as well as multimeric protein complexes, virus-like particles, membrane proteins, and proteins that are toxic to mammalian cells. Recently, BEVS has demonstrated particular utility in the commercial-scale manufacture of vaccines for human and veterinary applications.
Among the advantages of BEVS are the ability to express proteins with enhanced post-translational modifications compared to bacteria, lower costs of reagents and fewer equipment needs compared to mammalian expression, and a demonstrated record of scalability for the commercial production of vaccines and cell therapy reagents. 1
BEVS platforms, however, continue to possess a number of shortcomings compared to plasmid-based mammalian transient systems including: lot-to-lot variability of the expression media due to the presence of undefined components such as yeastolate, lower protein titers, and longer overall time to protein. These factors can significantly hamper product development at a time when manufacturers are under continual pressure to develop vaccines and other biologics more quickly and efficiently.
Unlike many mammalian expression systems that transitioned to chemically-defined media formulations more than a decade ago, insect expression systems have continued to rely on poorly defined, yeastolate-containing media formulations that can impart significant lot-to-lot variability on cell growth, baculovirus production, and protein expression.
Yeastolate-containing media are further limited in their capacity to sustain high-density cell growth and consequently support high-cell-density baculovirus infections, two critical aspects required to increase biomass and improve protein titers on a per volume basis.
Compared to mammalian transient protein expression, the time from gene to protein for insect systems is significantly longer, typically on the order of 3–4 weeks, due primarily to the time required to generate a baculovirus stock using adherent Sf9 cells followed by subsequent amplification of virus through P1, P2, and/or P3 stocks to obtain sufficient virus for protein expression runs.
To address the shortcomings of traditional insect expression systems, the ExpiSf™ Expression System was developed to eliminate the variability associated with yeastolate-containing culture medium, to increase protein titers, and to reduce total time to protein—all to streamline research such as vaccine development from bench to clinic.
Similar to the Expi293 and ExpiCHO mammalian expression systems, a systems-based approach was employed during development of the ExpiSf Expression System.
As an initial step in this process, the first chemically defined (CD), yeastolate-free insect cell culture medium, ExpiSf CD Medium, was developed to support high-density cell growth, high-titer virus production, and enhanced protein expression. Multiple lots of ExpiSf CD Medium were formulated and compared for cell growth and protein expression levels consistent growth kinetics (Figure 1A) and protein titers (Figure 1B) were obtained across four different lots of ExpiSf CD Medium.
Next, Gibco Sf9 cells were adapted into ExpiSf CD Medium through extensive long-term passaging to generate the high-density ExpiSf9 cell line. ExpiSf9 cell growth characteristics were compared to traditional Sf9 cells by culturing Sf9 cells in various yeastolate-containing media for at least 10 passages to ensure adaptation.
Compared to Sf9 cells grown in yeastolate-containing media, ExpiSf9 cells grown in ExpiSf CD Medium achieved higher peak viable cell densities in standard shake flask cultures (>20 × 10 6 cells/mL vs. 2–10 × 10 6 cells/mL Figure 1C), approximately double the peak density of the next highest density media formulation. ExpiSf9 cells also possessed a broad log-phase growth range spanning from approximately 4–12 × 10 6 viable cells/mL, with a stable doubling time of
Taking advantage of the higher densities that ExpiSf9 cells achieve in ExpiSf CD Medium, an expression enhancer (ExpiSf Enhancer) was developed to work in conjunction with the ExpiSf CD Medium to allow for consistent, high-density infection of ExpiSf9 cells at 5 × 10 6 cells/mL, leading to multifold improvements in protein titers compared to traditional Sf9 workflows, in which cells are infected at 1–2 × 10 6 cells/mL in yeastolate-containing media (Figure 1D).
Since optimal baculovirus infection of Sf9 cells is thought to occur when cells are in log-phase growth, the extended log-phase growth range of the ExpiSf9 cells, in conjunction with the ExpiSf Enhancer, allows for ExpiSf9 cells to be infected at significantly higher densities than traditional insect workflows, thereby increasing protein titers on a per volume basis.
Lastly, to reduce overall time to protein, the ExpiFectamine™ Sf Transfection Reagent was developed to allow for gentle, nontoxic transfection of high-density ExpiSf9 suspension cultures with large bacmid DNA. Incorporating suspension-based transfection for baculovirus generation enabled easy preparation of 100 mL (or greater) of high titer (1–5 × 10 9 infectious virus particles/mL), high-quality P0 virus, completely eliminating the need for additional virus amplification for typical liter and subliter workflows.
Additionally, suspension-based bacmid transfection shortens overall time to protein by up to 50% (Figure 1E) by eliminating the need for virus amplification while at the same time removing the risk of deleterious virus passaging effects (whereby gene incorporation/protein expression levels decrease during generation of P1 + virus stocks), ensuring that the highest quality baculovirus is used for protein expression runs.
Figure 1. Performance characteristics of the ExpiSf Expression System. (A) Four different lots of ExpiSf CD Medium demonstrated consistent growth of ExpiSf9 cells with peak viable cell densities (VCDs) of
20 × 106 cells/mL. (B) Green fluorescent protein (GFP) titers were 4–5-fold higher in all lots of ExpiSf CD Medium tested compared to a traditional Sf9 workflow using yeastolate-containing (YC) medium. (C) ExpiSf9 cells in ExpiSf CD Medium exhibited superior cell growth compared to five yeastolate-containing insect cell media. (D) ExpiSf Enhancer, used in conjunction with ExpiSf CD Medium and ExpiSf9 cells, generated 3-fold higher GFP titers than a traditional Sf9 workflow ExpiSf Enhancer nearly doubled protein titers compared to the ExpiSf System without enhancer addition. (E) The ExpiSf Expression System reduced the time required to go from bacmid DNA to protein expression by half by eliminating the need for virus amplification via direct generation of high-volume and high-titer P0 virus stocks.
Comparison of ExpiSf to Traditional Sf9-Based Workflows
The expression levels of three different proteins—green fluorescence protein, human Fc fusion protein, and tumor necrosis factor-alpha (TNF-a)—were compared between the ExpiSf system and a traditional Sf9 workflow in which cells are infected at 1–2 × 10 6 cells/mL in yeastolate-containing media.
Compared to five different yeastolate-containing media tested using traditional Sf9 workflows, the high-density ExpiSf Expression System generated 3–5-fold improvements in expression levels across the three proteins tested (Figure 2A).
G-protein-coupled receptors are commonly expressed in insect cells, in part because of the potential for toxicity in mammalian cells, as well as the desire for reduced glycosylation for structural biology studies. Cannabinoid receptor 2 (CB2) was expressed in the ExpiSf system and in a traditional Sf9 workflow.
Optimal cell harvest time was determined to be 48 hours post infection in the ExpiSf corresponding to a cell viability of 70% at the time of harvest longer infection times led to decreased viability without improving CB2 expression per cell (Figures 2B & 2C). CB2 expression (as measured by total number of CB2 molecules per cell by quantitative flow cytometry) was 10-fold higher in the ExpiSf Expression System compared to the traditional Sf9 workflow (Figure 2D).
This improvement was due to both per cell increases in expression levels as well as the significantly higher density of ExpiSf9 cells in a given volume compared to the traditional Sf9 workflow.
Figure 2. Comparison of the ExpiSf Expression System to traditional Sf9 workflows using various yeastolate-containing media. (A) Expression levels of an Fc fusion protein, green fluorescent protein (GFP), and tumor necrosis factor-alpha (TNF-a) were on average >4, >3, or >5-fold higher in the ExpiSf expression system than those obtained using various yeastolate-containing media in a traditional Sf9 workflow. (B) Optimization of CB2 G-protein-coupled chemokine receptor harvest time and (C) post-infection viability kinetics in the ExpiSf Expression System. (D) Increased total CB2 expression levels obtained in the ExpiSf Expression System compared to traditional Sf9 workflow in Sf-900 II medium increased expression of CB2 is due to both higher per cell expression as well as greater cell density in a given volume for the ExpiSf Expression System.
Glycosylation Patterns and Protein Functionality
Although high protein yields are desirable, resultant proteins are less valuable if they are aggregated, misfolded, degraded, or improperly glycosylated. The quality of the proteins expressed in the ExpiSf system was compared to the quality of the same proteins expressed using a traditional Sf9 workflow with yeastolate-containing medium.
Using secreted alkaline phosphatase (SEAP) as a model protein, glycosylation patterns generated in the ExpiSf were shown to be highly comparable to those of a traditional Sf9 insect workflow, with the predominate glycoforms being Man3, Man3F, and Man6 in both instances (Figure 3A). SDS-PAGE showed a single band with a molecular weight of
57 kD for both workflows (Figure 3B).
The biological activity of TNF-α expressed in the ExpiSf system and by traditional Sf9 workflow was assessed using an NF-κB luciferase reporter gene assay. HIS-tagged TNF-a was expressed and purified by Ni-NTA, and its concentration was determined by A280. TNF-a was expressed at >4-fold higher levels in ExpiSf compared to the traditional Sf9 workflow (Figure 3C) reporter gene assay results demonstrated equivalent biological activity (relative luminescence units RLUs) for the proteins, respectively (Figure 3D).
In summary, the ExpiSf Expression System represents a significant advance in insect cell protein expression in terms of media consistency, protein yield, and time. It enables researchers to streamline protein expression and vaccine development to shorten time lines from bench to clinic.
Figure 3. Comparison of glycosylation patterns and biological activity for proteins expressed in the ExpiSf Expression System and by traditional Sf9 workflow. (A) Glycosylation patterns of secreted alkaline phosphatase (SEAP) were highly similar in the ExpiSf Expression System and in a traditional insect workflow using Sf900-II medium. (B) SDS-PAGE of SEAP purified from the ExpiSf Expression System and a traditional Sf9 workflow using Sf900-II medium. (C) The ExpiSf Expression System generated >4-fold higher TNF-a expression levels compared to a traditional Sf9 workflow. (D) TNF-a activity, as measured by luciferase-based NF?B reporter gene assay, showed equivalent biological response for protein generated in the ExpiSf Expression System and by traditional Sf9 workflow.
We are able to produce and purify the proteins you want in multi-milligram quantities, and our expertise in protein characterisation means that you can have confidence that we will deliver you high quality protein that has passed our rigorous quality control process.
Protein Science Services
We can offer you a complete protein science solution, including:
- Construct design
- Protein expression services and expression system optimisation using E. coli, insect cells, or mammalian cells
- Protein production services
- Purification by affinity, ion-exchange and size-exclusion chromatography
- Protein characterisation
Combinatorial Domain Hunting
Often our scientists will be able to use literature-informed or bioinformatics-based approaches for expression construct design. But if you already know that these methods have been unsuccessful, or you think they will not work on your protein, our unique proprietary technology Combinatorial Domain Hunting (CDH) allows us to quickly identify soluble, highly expressible protein constructs for the most problematic drug target proteins.
The high quality of the proteins that we produce means that you can use them to support a number of key processes in drug discovery and naturally our protein production capability pipelines smoothly into other Domainex services such as:
An exciting recent development at Domainex is our PoLiPa technology for making pure and stable preparations of solubilised membrane proteins such as GPCRs. The PoLiPa approach seems set to revolutionise membrane protein drug discovery, making these otherwise difficult targets accessible to structural biology, biophysical assays, and other screening technologies.
How is a gene structured?
The genome is not simply a chain of protein-coding genes one after the other. Even within one gene, the protein-coding sequences are interrupted by non-coding regions. These non-coding interruptions are known as intervening sequences or introns. Conversely, the coding sequences that are actually expressed are called exons.
Most, but not all structural eukaryote genes contain introns. Importantly, these introns are initially transcribed with the exons to form the pre-mRNA, however, they are cut out of the transcript and the remaining exons are joined together before the mRNA is finished being processed. This process is called RNA splicing. This completed, processed mRNA is called the mature mRNA.
Generally, the more complex organisms have more and larger introns. One reason for the existence of the intron/exon structure is that exons can code for different functional regions of proteins, so with the inclusion/exclusion of certain exons, genes can produce various forms of their protein for in different tissues or at different times. This is because the transcription machinery can skip certain exons and include other ones, creating transcripts with different sequences. This process is called alternative splicing, and represents an important layer of controlling the proteins that are produced in cells.
Genes encode proteins and proteins dictate cell function. Therefore, the combination of genes that are active in a particular cell determine the identity of the cell and the tasks it is carrying out. The activation of genes is called gene expression, and it is tightly regulated at several points, from the initial process of transcription, to splicing, to the translation of the proteins and the modifications to the final protein structures. These processes are tightly controlled at every step to closely monitor and maintain cell types with the required characteristics.
How is gene expression regulated?
The main control point for gene expression is usually at the start of transcription. That is, controlling the signal that tells the cell to produce this mRNA and make this protein.
Transcript processing provides an additional level of regulation. This level of regulation includes splicing, where alternative transcripts can be produced depending on the needs of the cell. Additionally, newly synthesized transcripts can be enzymatically broken down to control protein levels in the cell in response to different cues.
The variety of cell types that exist in a multicellular organism comes from the complexity brought about by variety of potential gene expression profiles. Different cell types possess distinct sets of regulators that initiate or repress the production of different transcripts and proteins
As mentioned previously, a large proportion of the genome codes for important regulators. That is, the sequences do not make a protein, but the sequences are key in modulating the expression of other genes.
Transcription usually starts when RNA polymerase binds to an important regulatory region called the gene promoter. This sequence is usually located just upstream from the starting point for transcription. The binding of specific proteins to the promoter is usually required for the gene to be transcribed. Thus, the cell can regulate whether the gene is expressed or not through these regulatory factors, which can be general or cell-type specific.
More recently, another type of regulatory elements have been discovered, called enhancer sequences. These also represent a binding site for other regulatory proteins, and they help to control and fine-tune the expression of a gene. There is no strict criteria for what defines an enhancer region, and the landscape of enhancers in the genome is hugely complicated. Enhancers can also only be active in certain cell types of conditions, or can be active all the time. One interesting feature of enhancers is they can be located thousands of nucleotides away from the gene they control. Further complicating things, some regulatory elements or proteins can affect the transcription of multiple genes, and some regulatory proteins can even have different roles for different genes!
The timed turning off/on of genes in a cell represents on layer of control of the content of proteins in a cell, and thus the identity of that cell. The complexity of the genome in eukaryotes is due to the presence of multiple regulators on the DNA. The concerted actions of regulatory sequences in the DNA, the presence of regulatory factors, and the post-transcriptional/translational processing of a transcript or a protein allows fine-tuning of the gene expression patterns in a cell. This allows cells to perform their required tasks and to respond quickly to changes in the environment.