We are searching data for your request:
Upon completion, a link will appear to access the found materials.
I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with both genes in it into the chromosomes. My questions are:
- If I use the same promoter (CMV, SV40 or an inducible one) for both genes how would this affect the expression levels? I read in some papers that one of the genes might get expressed lower than the other. If you have first-hand experience I will be grateful if you share it.
- If I use the same promoter for both, but put them far away (i.e. ends of the cassette) would the expression levels get affected again? It sounds simple enough but I think people would have published something if they had a good result with that.
- If I use different promoters for each gene (e.g. CMV for one and Tet-inducible for the other) how would it work? I couldn't find a paper which used this and I would like to continue with this approach; therefore, if you have any experience I will be really grateful for any help.
I don't want to use IRES or 2A-peptide. I would like to be able to control this experiment in transcription stage.
You can use a bidirectional promoter. The problem that you mentioned about proteins not expressed in same level happens because of competition for polymerase. But there are well optimized parts and also commercially available vectors that work fine.
You can clone genes in a serial order. It won't be a problem. Just leave a 100bp linker after the polyA signal of previous gene. If your cassette becomes too huge, then insertion becomes slightly difficult. That's why people use IRES or TA-peptides, etc.
Retroviral based insertions work quite decently, but you can't control copy number variation between different cells.
You can use two different promoters. The dynamics will depend on the promoter strength and concentration of inducer.
I was going through my questions and realized this is still unanswered. Well, I have the answer now.
I designed the construct with two separate promoters (CMV for one and Tet-inducible for the other) and got really good results. I haven't had any difficulties in expressing the proteins. I was able to control the Tet-inducible one very nicely and got steady levels of the other protein with CMV.
Long story short: you can safely put two genes under two separate promoters, prepare stable cell lines with them and get good expression levels.
What is the best way to express two proteins in a mammalian cell? - Biology
What is recombinant protein? Proteins are one of the most important biological molecules for life. And these proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli and transporting molecules from one location to another. However, when we want to analyze the structure, function, mechanism, pathway or other facts of these proteins, we will find that the quantities are not sufficient from nature source. In that case we need to produce high quantity and purity proteins in a short time and low cost in our lab. Thus, recombinant protein technology is required. Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. Recombinant protein is encoded by recombinant DNA, which has been cloned in a system that supports expression of the gene and translation of mRNA. The recombinant DNA, usually the cDNA sequence of the target protein, is designed to be under the control of a well characterized promoter and express the target protein within the chosen host cell to achieve high-level protein expression. Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein or a large quantity of protein.
Choosing an appropriate protein expression system is the key to the success of recombinant protein expression. Several factors need to be taken into consideration, including target protein property, intended application, protein yield and cost. Furthermore, challenges exist for many protein expression projects, especially for large protein, membrane protein, nuclear protein and proteins with heavy post-translational modifications.
Nowadays, there are several expression systems for use. Different systems have different features and applications. Here, we will introduce four systems which are commonly used in research and industrial. They are bacteria expression system, yeast expression system, baculovirus expression system and mammalian expression system.
Fig1. Four Expression Systems
E.coli expression system is the primary use and most mature expression system. The main method is transfer a vector which inserted target DNA fragment to host cell. And then induce protein expression by IPTG.
As the earliest development and the most widely used classical expression system, the E. coli expression system has the advantages of clear genetic background, fast breeding, low cost, high expression, easy purification of the product, good stability, strong anti-pollution ability and wide application range. However, there are many shortcomings in the prokaryotic expression system: not all proteins are soluble. Incorrectly folded proteins formed in cytoplasm can form insoluble aggregates called inclusion bodies, which leads to the difficult purification. Moreover, the post-translational modification processing of the prokaryotic expression system is imperfect and the biological activity of the expressed product is low.
Thus, other more sophisticated systems are also being developed such systems may allow for the expression of proteins previously thought impossible in E.coli, for example, glycosylated proteins.
Fig2. Incorrectly folded and inclusion bodies
Yeast expression system as a new exogenous protein expression system, contained merits from both prokaryotic and eukaryotic expression system. It is being widely used in the field of genetic engineering. Complete gene sequence of Saccharomyces cerevisiae are sequenced in 1996. The use of S.cerevisiae in the brewing and bread industry has been known for thousands of years and is considered to be GRAS (generally recognized as safe) creatures that do not produce toxins and has also been recognized by FDA. Thus, the productions expressed from yeast system need not a lot of host safety experiments.
However, compared with the new yeast system, S.cerevisiae expression system is not suitable for high-density culture lacking strong and strict regulation promoter. And the secretion efficiency is low. Especially, many target protein which molecular weight greater than 30kD almost does not secrete.
Later, people have developed fission yeast and methanol yeast expression system. Among them, the methanol yeast expression system is the most widely used yeast expression system. Nowadays, mainly used methanol yeasts are H Polymorpha, Candida Bodini and Pichia Pastris. Pichia Pastoris is the most popularity tool. Most of the methanol yeast contains the methanolic yeast oxidase gene-1 (AOX1). The exogenous gene was expressed under the action of the promoter (PAOX1). PAOX1 is a strong promoter using glucose or glycerol as carbon source. The expression of AOX1 gene in methanol yeast was usually inhibited. And PAOX1 could be activated when methanol was the sole carbon source. Therefore, the expression of AOX1 gene could be increased under the control of the gene. The use of methanol yeast to express exogenous protein production is often up to grams. Compared with S.cerevisiae, its translation is closer to mammalian cells and does not undergo hyperglycosylation.
The insect expression system is a widely used eukaryotic expression system that has the ability to translate and modify foreign proteins similar to higher eukaryotes. The expression of exogenous protein in the insect cell system by recombinant baculovirus is a more popular expression method. The protein level is up to 1
500mg / L. But it is restricted and influenced by many factors such as culture medium, oxygen supply and logarithmic growth and so on.
Baculovirus is the largest group of known insect viruses and is the earliest, most studied and applicable insect virus. The baculovirus genome is a single closed circular double-stranded DNA molecule with a size of 80-160 kb. Its genome can be replicated and transcribed in insect nuclei. DNA replication is assembled in the bark of the baculovirus, which has a great flexibility and could accommodate large fragments of foreign DNA insert. It is the ideal carrier for the expression of large fragments of DNA.
The main advantages of baculovirus system include:
- Recombinant protein has complete biological function, such as protein correct folding and disulfide bond
- Post-translation modification
- High level of expression, up to 50% of the total protein amount
- Accommodate large insert protein
- Simultaneously express multiple genes.
The main drawback is that exogenous protein expression is under the control of the very late viral promoter, where the cells begin to die due to viral infection.
Insect expression system is normally used for production of membrane proteins, although the glycosylations may be different from those found in vertebrates. In general, it is safer to use than mammalian virus, since it has limited host range and does not infect vertebrates without modifications.
Recombinant proteins expressed in mammalian cell commonly use plasmid transfection and viral vector infection. Stable cell line using plasmid transfection takes several weeks or even months, while the virus vector can quickly infect cells within a few days.
Depending on the temporal and spatial differences in protein expression, the expression system can be divided into transient, stable and induced expression systems. Transient expression system refers that host cell cultures without selection pressure and exogenous vector gradually lost while cell division. The target protein expression duration is short. The advantage of transient expression system is simple and short experimental period. Stable expression system means that the carrier DNA replicate and express long time stably in host cell. Due to the need of select resistance and pressure steps, stable expression is relatively time-consuming and laborious. Induction expression system refers that the target gene begins to express when induced by foreign small molecules. The use of heterologous promoters, enhancers and amplifiable genetic markers can increase protein production.
The mammalian expression system has unique advantage in protein initiation signals, processing, secretion, glycosylation and is suitable for expressing intact macromolecules.
Fig3.Stable cell line development
The foreign protein produced by mammalian cells, which is closer to the native protein, has much more activity than proteins produced by prokaryotic expression system or by other eukaryotic expression system, such as yeast and insect cells. The disadvantage of the technique is complicated, high requirement, low yield and sometimes viral infections exist.
Above all, there are advantages and disadvantages for all these expression systems. The advantage of E.coli and yeast expression system is high expression level and low cost. But their modification systems are different from insect cell and mammalian cells. Protein produced by mammalian cell is similar with native protein. However, the disadvantage is low expression level and complicated operation. The biological activity and immunogenicity of the recombinant proteins produced by different expression systems are sometimes different due to the diverted post-translational processing. Therefore, when considering which expression system to choice, we need take a variety of factors into consideration, such as whether the target protein has toxic, whether the protein need biological activity, whether the glycosylation is needed for the protein and cost efficiency, yield, purification, safety and so on. Supported by experienced experimental knowledge and consider all these factors, our expertise will make suitable determination and choice.
Table1. Simply Compare among Expression Systems
|Bacterial Expression System||Yeast Expression System||Insect Expression System||Mammalian Expression System|
(relative to human)
|Application||Prokaryotic protein, simple eukaryotic protein||Intracellular/Secreted Protein, Disulfide-bonded protein, Glycosylated protein||Membrane protein, Large-size protein, |
Viral vaccines, signaling proteins, cytokines, kinases
|Complex eukaryotic protein, Protein need accurate PTM|
We provide all protein expression system services in our company. If you have any questions on recombinant protein and expression system choice, welcome to contact us for details!
Normal life span and cell line derivation
During the early stages of development, animal cells undergo extensive proliferation and differentiation while developing into different tissues and organs. In an adult, the vast majority of cells are quiescent although they are metabolically active and perform their physiological roles, such as filtration in the kidneys or synthesis and chemical transformation in the liver, most are not actively dividing. Most normal adult cells only divide in response to stimuli to replenish old or damaged cells. Only cells in specific tissues, such as skin or epithelial intestinal cells, divide regularly.
The body has over 200 different types of cells, many of which cannot be excised and grown in culture. Cells that are more amenable to culture include fibroblasts and certain epithelial cells. A first step in cell isolation is to explant a tissue in a physical and chemical environment suitable for those cells to survive and proliferate.
A permissive environment for cell growth requires a complex mixture of nutrients, including sugars, amino acids, vitamins, minerals, and growth factors such as insulin. Except for certain cell types in blood, cells derived from tissues are anchorage-dependent, meaning they do not grow as free-floating individual cells. Therefore, after being released from the tissue environment, cells require a surface on which they can attach, otherwise they will fail to survive and divide.
After attachment, cells grow and expand onto empty surfaces until the entire surface is covered in a layer that is one cell thick (i.e., a monolayer). At this point, they stop dividing and reach a state called contact inhibition. Next, an enzyme, such as trypsin, is used to degrade the proteins that “glue” the cells to the surface, thereby releasing the cells into solution. Once detached, the cells can be transferred to a culture vessel with a larger surface area to resume growth.
This cycle of attachment, cell expansion, and detachment can repeat many times, with each cycle comprised of multiple cell divisions. However, most normal cells have an internal clock that counts their own doublings. Cell division stops once the so-called Hayflick limit is reached (3, 4). Most cells derived from tissues can divide up to 40–60 times before ceasing to proliferate (becoming senescent) and exhibiting abnormal appearance. Nevertheless, the number of doublings that these cells can sustain in culture is sufficient for vaccine production applications.
The senescence and contact inhibition exhibited by these cells are hallmarks of cells from normal tissues. Certain cells isolated from cancers, however, are immortal and can overcome contact inhibition. More than a half-century ago, scientists succeeded in isolating cells that survived senescence (5, 6). These cells continued to divide after all others died. Interestingly, unlike cancer cells, some of the surviving cells still obeyed contact inhibition and looked morphologically normal.
These immortal cells that bypass the Hayflick limit and continue to divide are called cell lines and are immortal in culture, unlike cell strains isolated from normal tissues. Cell strains and cell lines differ in another important way: All of the cells in cell strains have normal chromosomes with two sets per cell, while cells from cell lines do not typically have two sets of chromosomes, even if they are normal morphologically.
Many cell lines induce tumor formation when injected into immunocompromised mice. However, because they can be cultured forever, cell lines can be genetically engineered to produce a product in virtually unlimited quantities. For this reason, all of the therapeutic proteins produced in mammalian cells employ cell lines.
Transient transfection methods
Liposomes are synthetic analogues of the phospholipid bilayer, the building block of the cellular membrane. These transfection compounds share a number of characteristics with their natural counterparts, including the presence of hydrophobic and hydrophilic regions of each molecule which allow for the formation of spheroid liposomes under aqueous conditions. In the presence of free DNA or RNA, liposomes encapsulate the nucleic acids to create an efficient delivery system. The charge, composition and structure of the liposome defines the affinity of the complex for the cellular membrane. Under specific conditions, the liposome-nucleic acid complex is able to interact with the cell membrane to gain access into the cell by endocytosis and subsequently release the nucleic acids into the cytoplasm. There are several factors that determine the successful delivery of nucleic acids by liposome into mammalian cells, including particle size, lipid formulation, charge ratio and the method of liposome preparation.
Alternatives to liposomes include non-liposomal lipids and polymers capable of forming complexes with nucleic acid to form micelles, or tiny encapsulating droplets. The transfection is usually performed under aqueous conditions, which enables the lipophilic portion of the amphiphilic compound, or the part of the droplet that displays affinity for fatty-acid compounds such as the cell membrane, to form the micelle capsule that encases the exogenous nucleic acids.
Dendrimers are highly branched, globular macromolecules that are capable of interacting with DNA to form small complexes. Dendrimers are stable in biological liquids and are not sensitive to temperature. These properties make dendrimers highly efficient tools for tissue culture transfections. The downside of dendrimers is that they are non-biodegradable and therefore may be toxic to cells with prolonged exposure.
Electroporation is a highly efficient technique for delivering exogenous nucleic acids to suspension cells and non-adherent primary cells (like lymphocytes). This technique uses electricity to create transient pores (electropores) in the cellular membrane to enable the uptake of charged nucleic acid molecules (RNA or DNA) into the target cells.
Positive selection in mammalian cells
To achieve stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA into the genome. For the purposes of this post, we will define positive selection as the means of picking up positive traits (i.e. the plasmid contains a cassette that will make cells resistant to a toxin), whereas negative selection would be the picking up of a negative trait (i.e. the plasmid contains a cassette that will make cells sensitive to a toxin). In the table below we focus on positive selection however, negative selection techniques can be used in conjunction with positive selection to ensure your gene gets targeted to a specific location within the genome.
Positive selection in mammalian cells works similarly to that in bacteria and a table of the most commonly used selection markers are listed below:
HeLa, NIH3T3, CHO, COS-1, 293HEK
*Not comprehensive. ** In eukaryotes. ***The concentration used for selection is typically more (double) than that used for maintenance of a transfected cell line.
Optimizing Protein Expression
Like a train that consists of a particular sequence of cars, from locomotive to caboose, proteins can only “leave the station” after each of its segments, amino acids, are hooked together.
Actually, the shuttling of cars in a rail yard is a lot simpler than the processes that make up protein expression—processes that begin with transcription, culminate in translation, and are even elaborated in post-translational modification. It is possible, however, to take control of these processes and freight valuable cargo such as complex biopharmaceuticals.
Aiming to provide practical solutions for today’s challenges, the recent Bioprocessing Summit focused one segment of its conference program to advances in protein expression. This segment covered new approaches such as utilizing genome-scale RNA interference (RNAi), improving glycoengineering of the ever-popular Chinese hamster ovary (CHO) cell expression systems, and harmonizing transient and stable expression to enhance drug development.
Presentations were also devoted to insect cells. Like other expression systems, insect cells can be used to produce biotherapeutics with careful optimization of manufacturing-compatible processes. Finally, some presentations touched on outsourcing issues, such as the relative advantages of using one or several companies for the multiple processes involved in protein expression.
Given the myriad challenges that can compromise efficient protein expression, it sometimes makes sense to consult specialists. For example, outsourcing issues have prompted many companies to work with Ingenza, which says it aims to be the partner of choice in industrial biotechnology and synthetic biology.
“There are many unseen as well as known facets to effective protein expression that require a high level of expertise to simultaneously optimize,” stated Ian Fotheringham, Ph.D., Ingenza’s managing director. “Some laboratories have knowledge in one aspect or another, but often it is more efficient to work with someone with the capabilities to address the multiple aspects of effective protein expression. We are distinctive in that respect.”
Dr. Fotheringham asserted that outsourcing is now a well-established option in the field of protein expression: “Customers want someone who can produce, innovate, troubleshoot, and continually dialog with them to increase their chances for success. We are a multidisciplinary company for a reason.”
As an example, Dr. Fotheringham described working with a leading U.S. biopharma company whose expressed target protein was truncated in the recombinant host. “First, we optimized the target gene for expression in Escherichia coli using a library of
50,000 coding region sequence variants. We devised a simple petri plate screen that rapidly identified the best full-length performers.
“Prompted by such successes, we subsequently provided GMP-compliant production strains for many of our customer’s targets. Other projects have successfully involved dual cassette vectors (such as target and chaperone), alternate hosts, novel induction strategies, and targeted genome integration to optimize recombinant protein stability, production, and purification.”
Ingenza also works with academic institutions and emerging spin-outs to translate innovative research. Dr. Fotheringham cited the example of epidermicin, a potent antimicrobial that was discovered by one of the company’s university partners. Epidermicin can combat methicillin-resistant Staphylococcus aureus (MRSA) infection, but initially it was expressible only in low amounts due to host toxicity.
“We utilized our proprietary inABLE ® combinatorial assembly platform to prepare and screen cleavable fusion constructs which, combined with design-of-experiment (DOE)-driven fermentation, permitted over-production of epidermicin and its derivatives,” detailed Dr. Fotheringham. “Success is enabling a university spin-off to exploit the outcome.”
The company is now moving further into the GMP biologics arena with key clients, using optimized microbial and mammalian production systems and modular single-use fermentation approaches to provide drug substances for clinical evaluation and manufacturing.
Ingenza utilizes simple petri dish screens to optimize target gene expression in Escherichia coli using a library of
Harmonizing Transient and Stable CHO Expression
For those working in-house, expression of protein products in CHO cells remains one of the most popular expression platforms for a variety of reasons, said Yashas Rajendra, Ph.D., research scientist, Eli Lilly and Company. “CHO systems,” he explained, “are inherently adaptable and easy to scale up to industrial bioreactors”
However, for early-phase drug discovery of complex biopharmaceuticals, companies often rely on HEK293 transient expression platforms. “This can increase the risk of surprises when molecules are advanced and eventually expressed in CHO,” warned Dr. Rajendra. “To reduce this risk, we developed a transient CHO platform that is based on the same cell line, media package, and DNA expression cassette used for our stable CHO platform.”
According to Dr. Rajendra, harmonization of transient and stable expression approaches can provide better predictability of protein quality and expression when transitioning the molecules from discovery to development and ultimately manufacturing. “This transient CHO system can rapidly (within seven days) generate high titers,” asserted Dr. Rajendra. “The system is scalable to 10 L.”
Dr. Rajendra’s group introduced another modification: the use of stable CHO pools (instead of master wells or clones) to generate gram quantities of therapeutic protein. “It takes 2–4 weeks to generate a stable CHO pool,” noted Dr. Rajendra. “In contrast, it usually takes several months to generate clonal CHO cell lines. Hence, speed is one major advantage of stable pools.
“Additionally, advantages over transient expression include use of smaller amounts of plasmid DNA for transfection, ease of volumetric scale-up, and the flexibility of performing multiple production runs over an extended period of time using a frozen cell bank. We recently published CHO pool titers ranging from 2 to 7.6 g/L.”
Dr. Rajendra indicated that at present, stable pools are primarily used for preclinical material generation due to concerns of clonal heterogeneity, genetic and expression stability, and product quality consistency. In the future, however, the use of stable pools may extend to the generation of toxicology lots and the first human dose studies.
“Although this is going to take some time,” Dr. Rajendra concluded, “given the advancements in host cell engineering, transposon-mediated approaches, and analytical tools that allow for in-depth product quality assessment, I am hopeful that we will see a shift from the ‘clonality’ paradigm to a “product quality consistency’ paradigm.”
Glycoengineering in CHO
Although they are widely used, CHO expression systems present challenges similar to those posed by other mammalian cells. In particular, CHO cells produce recombinant proteins that are heterogeneous in complex-type N-glycans.
“Glycosylation is one the most important post-translational modifications of proteins,” stated Andrew (Cheng-Yu) Chung, researcher, department of chemical and biomolecular engineering, Johns Hopkins University (JHU). “Glycans on proteins play a key role, particularly in protein folding. Thus, they significantly impact protein stability and protein-protein interactions.”
Chung, who is in the laboratory of Michael J. Betenbaugh, Ph.D., is studying sialylation of engineered proteins. “Because of the negative charge, size, and hydrophilic characteristics of sialic acid groups, sialylation is one of the very critical modifications on the glycan terminus,” Chung explained. These properties allowed sialic acid to have a substantial influence on protein-protein interactions. Further, sialylation can affect biotherapeutics’ efficiencies.”
Chung and colleagues examined the application of genetic engineering strategies to modulate the level of sialylation content on various potential biotherapeutics. “One common way to manipulate the glycan structure is via genetic engineering tools such as CRISPR/Cas9 to knockin/knockout or overexpress certain glycoenzymes,” he noted. “For example, overexpressing glycoenzymes involved in the sialylation pathway is a strategy to produce biologics with increased sialic acid content that may affect circulation retention time (CRT). Increased CRT may mean lower doses for patients.”
Another strategy to make glycosylation in CHO cells more uniform and similar to the analogous process in human cells is to add specific chemical supplements to the cell culture medium.
Chung’s group decided to try both strategies—genetic engineering and the addition media supplements—at the same time. “The genetic engineering of CHO cells can be used to produce biologics with enhanced sialylation as well as higher protein titers,” Chung indicated. This approach, continued Chung, has been combined with the use of chemical supplements. The specific chemical analogs that are added to the feed were developed by Kevin Yarema, Ph.D, an associated professor of biomedical engineering at JHU.
Insect Cell Expression
While most recombinant biotherapeutics are produced in mammalian cells, others are produced in insect cells. For example, an insect cell platform was selected to express recombinant proteins for Genocea Biosciences.
“Our therapeutic vaccine (GEN-003) against genital herpes is comprised of two recombinant proteins combined with an adjuvant,” said Rajiv Gangurde, Ph.D., associate director of protein production. “Both proteins are expressed in insect cells.
“The choice of the expression platform was primarily driven by specific post-translational modification(s) required for the activity, that is, the ability to elicit an appropriate immunogenic response, by one of the antigens. These modifications are not just specific to the host cells in which the recombinant protein is expressed, but are also well-tolerated in humans, which is one of the biggest advantages of using insect cells.”
Genital herpes is a serious chronic infection affecting more than 400 million people worldwide and over 50 million in the United States alone. Dr. Rajiv pointed out that there has been no real innovation on behalf of patients for decades, not since oral antivirals were approved.
Prior to expressing the product in insect cells, Dr. Rajiv and colleagues needed to overcome some specific hurdles. “Early on, we faced several challenges in developing manufacturing-compatible processes,” he noted. “We have overcome those hurdles through extensive in-house process development work and choosing a contract manufacturing organization with insect cell production scale-up experience.
“One protein is expressed as a soluble, secreted protein, while the other is an intracellular, insoluble protein. This fundamental difference in expression makes it easier to express the proteins separately. Moreover, there are notable differences in the levels of expression hence, independent expression allows the required flexibility in downstream processing and drug product manufacture.”
The company, which is conducting its third clinical trial with GEN-003, expects to start Phase III in the second half of next year. Next, the company plans to file a Biologics License Application with the FDA in 2019. If the application is approved, the company will launch GEN-003 in 2020.
“We believe we have established GEN-003 as a highly attractive product that requires only a once-yearly injection offering similar efficacy to that of daily oral antiviral therapy,” asserts Dr. Rajiv. “Given that most patients choose not to take daily oral anti-viral pills and accounting for compliance challenges with daily therapy, we believe the real-world efficacy of GEN-003 from an annual injection could translate into significant patient benefits and a revenue opportunity in excess of $1 billion in the United States alone.”
Genocea Biosciences’ Rajiv Gurde, Ph.D., utilizes insect cells to separately express two key recombinant proteins for the company’s therapeutic vaccine, GEN-003, against genital herpes.
RNAi to Enhance Expression
Aside from its traditional use as a workhorse technique for basic research, RNAi also has applications for improving protein expression.
“RNAi has been often applied in the fields of medical and basic research, especially to understand disease mechanisms,” reports Joseph Shiloach, Ph.D., director of the Biotechnology Core Laboratory, Intramural Research at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. “We decided to use it for identifying genes that are currently not known to be involved in protein expression, yet whose knockdown could improve recombinant protein production in mammalian cells.”
Dr. Shiloach and colleagues performed a genome-scale, high-throughput RNAi screen using HEK293 cells expressing firefly luciferase. “The basic idea was to individually knock down approximately 22,000 genes one at a time and look for those that affected expression, but did not affect cell growth,” explained Dr. Shiloach. “We utilized three different siRNAs for each gene and performed the work collaborating with the National Center for Advanced Translational Science.”
Among the “hits” identified was the gene for ornithine decarboxylase antizyme 1. The protein product helps regulate intracellular polyamines that are needed for cell growth and proliferation. “We also performed a detailed investigation to confirm the findings,” continued Dr. Shiloach. “We are now assessing the long-term effects of knockdown, the mechanisms involved in metabolism, and feasibility of extending this approach to other mammalian cells.”
According to Dr. Shiloach, the take-home message from these studies is that genome-scale RNAi screens can identify key pathways and genes whose modulation may help improve the expression and production of recombinant proteins. “These studies are more a beginning than an ending,” he stated. “But we have established a different foundation for the targeted design of more efficient mammalian cell expression platforms.”
Expression levels of recombinant proteins (simple proteins as well as complex biologics) can be unpredictable in mammalian cells, and this causes major headaches for target discovery and scale-up manufacture, according to Tom Payne, Ph.D., head of cell engineering at Oxford Genetics. Scientists are familiar with finding that sometimes proteins express at unexpectedly low levels, even when under the control of strong promoters (e.g., CMV).
“To minimize protein-to-protein variability and maximize yield, we adopted a high-throughput approach to screen and optimize >5,000 recombinant promoters by random shuffling, >30 5’ UTRs in different configurations and >15 poly-A signals,” said Dr. Payne. “We also developed Kozak and stop codon libraries and screened >1,000 genes from diverse organisms encoding putative ancillary protein to generate expression plasmids that consistently produce high yields of diverse proteins in HEK-293 and CHO systems, termed SnapFast Pro.”
Validating the System
To validate this vector system, Dr. Payne and his team compared protein yields to an industry-standard protein-expression vector. To maximize the relevance of the dataset they algorithmically determined the frequency with which each gene in the human genome had been cited in publications (PubMed hits, 2000–2016), identifying those genes consistently increasing in hits, as an indication of importance in biomedical research.
“The top 150 genes were batch codon-optimized, FLAG-tagged, and sub-cloned into the expression vector or SnapFast Pro,” continued Dr. Payne. “Following transient transfection into either CHO-K1 or HEK-293 cells, lysates and/or supernatants (depending on whether the protein is secreted) were subjected to high-throughput automated western blot. For both secreted and nonsecreted proteins, SnapFast Pro gave consistently high-level protein expression, in many cases showing high expression where no protein could be detected with the reference expression vector.”
Oxford Genetics is a specialist synthetic biology company focused on providing DNA, protein, virus, and cell-line design and development solutions. Two of the firm’s scientists can be seen working on a custom cell-line development project looking to optimize expression of a client’s gene of interest.
Protein Purification Strategies
Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. The optimal approach often must be determined empirically.
The best protein purification protocol depends not only on the protein being purified but also on many other factors such as the cell used to express the recombinant protein (e.g., prokaryotic versus eukaryotic cells). Escherichia coli remains the first choice of many researchers for producing recombinant proteins due to ease of use, rapid cell growth and low cost of culturing. Proteins expressed in E. coli can be purified in relatively high quantities, but these proteins, especially eukaryotic proteins, may not exhibit proper protein activity or folding. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. 1996). However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates.
To simplify purification, affinity purification tags can be fused to a recombinant protein of interest (Nilsson et al. 1997). Common fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached (Stevens et al. 2001). Using expression vectors that include a fusion tag facilitates recombinant protein purification.
Isolation of Protein Complexes
A major objective in proteomics is the elucidation of protein function and organization of the complex networks that are responsible for key cellular processes. Analysis of protein:protein interactions can provide valuable insight into the cell signaling cascades involved in these processes, and analysis of protein:nucleic acid interactions often reveals important information about biological processes such as mRNA regulation, chromosomal remodeling and transcription. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a gene’s promoter, and interacting with other proteins in the nucleus. This regulation is required for cell viability, differentiation and growth (Mankan et al. 2009 Gosh et al. 1998).
Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. 2004 Hall et al. 2007 Hudson and Snyder, 2006). Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface. Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions. Immobilized proteins also can be used in protein pull-down assays to isolate protein binding partners in vivo (mammalian cells) or in vitro. Other downstream applications such as mass spectrometry do not require protein immobilization to identify protein partners and individual components of protein complexes.
Commonly used protein production systems include those derived from bacteria,  yeast,   baculovirus/insect,  mammalian cells,   and more recently filamentous fungi such as Myceliophthora thermophila.  When biopharmaceuticals are produced with one of these systems, process-related impurities termed host cell proteins also arrive in the final product in trace amounts. 
Cell-based systems Edit
The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level".   Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype.  
There are many ways to introduce foreign DNA to a cell for expression, and many different host cells may be used for expression — each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the host and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria (such as E.coli, B. subtilis), yeast (such as S.cerevisiae  ) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses (such as baculovirus, retrovirus, adenovirus), plasmids, artificial chromosomes and bacteriophage (such as lambda). The best expression system depends on the gene involved, for example the Saccharomyces cerevisiae is often preferred for proteins that require significant posttranslational modification. Insect or mammal cell lines are used when human-like splicing of mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.
Because bacteria are prokaryotes, they are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. Hence, multi-domain eukaryotic proteins expressed in bacteria often are non-functional. Also, many proteins become insoluble as inclusion bodies that are difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding.
To address these concerns, expressions systems using multiple eukaryotic cells were developed for applications requiring the proteins be conformed as in, or closer to eukaryotic organisms: cells of plants (i.e. tobacco), of insects or mammalians (i.e. bovines) are transfected with genes and cultured in suspension and even as tissues or whole organisms, to produce fully folded proteins. Mammalian in vivo expression systems have however low yield and other limitations (time-consuming, toxicity to host cells. ). To combine the high yield/productivity and scalable protein features of bacteria and yeast, and advanced epigenetic features of plants, insects and mammalians systems, other protein production systems are developed using unicellular eukaryotes (i.e. non-pathogenic 'Leishmania' cells).
Bacterial systems Edit
Escherichia coli Edit
E. coli is one of the most widely used expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription.
For example, a DNA sequence for a protein of interest could be cloned or subcloned into a high copy-number plasmid containing the lac (often LacUV5) promoter, which is then transformed into the bacterium E. coli. Addition of IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest.
E. coli strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack lon and OmpT proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides T7 RNA polymerase (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead. 
Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine,  components of human food, animal feed and pharmaceutical products.
Expression of functionally active human epidermal growth factor has been done in C. glutamicum,  thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general, secretory pathway (Sec) or the twin-arginine translocation pathway (Tat). 
Unlike gram-negative bacteria, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.
Pseudomonas fluorescens Edit
The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens, is used for high level production of recombinant proteins commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein. 
Eukaryotic systems Edit
Expression systems using either S. cerevisiae or Pichia pastoris allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media of proteins.
Filamentous fungi Edit
Filamentous fungi, especially Aspergillus and Trichoderma, but also more recently Myceliophthora thermophila C1  have been developed into expression platforms for screening and production of diverse industrial enzymes. The expression system C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media.
Baculovirus-infected cells Edit
Baculovirus-infected insect cells  (Sf9, Sf21, High Five strains) or mammalian cells  (HeLa, HEK 293) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.  It is useful for production of proteins in high quantity. Genes are not expressed continuously because infected host cells eventually lyse and die during each infection cycle. 
Non-lytic insect cell expression Edit
Non-lytic insect cell expression is an alternative to the lytic baculovirus expression system. In non-lytic expression, vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent gene expression.   This is followed by selection and screening of recombinant clones.  The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression.  Cell lines used for this system include: Sf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni cells, and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells.   With this system, cells do not lyse and several cultivation modes can be used.  Additionally, protein production runs are reproducible.   This system gives a homogeneous product.  A drawback of this system is the requirement of an additional screening step for selecting viable clones. 
Leishmania tarentolae (cannot infect mammals) expression systems allow stable and lasting production of proteins at high yield, in chemically defined media. Produced proteins exhibit fully eukaryotic post-translational modifications, including glycosylation and disulfide bond formation. [ citation needed ]
Mammalian systems Edit
The most common mammalian expression systems are Chinese Hamster ovary (CHO) and Human embryonic kidney (HEK) cells.   
-  myeloma lymphoblstoid (e.g. NS0 cell) 
- Fully Human
- Human embryonic kidney cells (HEK-293) 
- Human embryonic retinal cells (Crucell's Per.C6) 
- Human amniocyte cells (Glycotope and CEVEC)
Cell-free systems Edit
Cell-free production of proteins is performed in vitro using purified RNA polymerase, ribosomes, tRNA and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system. Due to the low expression levels and high cost of cell-free systems, cell-based systems are more widely used. 
GC and VS designed the experiments. GC, VS and MG performed the experiments. AP developed the models and AP and DdB conducted simulations. AP, GC and DdB drafted the manuscript. DdB and MdB conceived and supervised the collaboration and overall strategy of the project and edited the manuscript. All authors have read and approved the final manuscript.
Giulia Cuccato, Athanasios Polynikis contributed equally to this work.
Customer Testimonial for our Protein Expression & Purification Services
"GenScript provides fast, professional protein synthesis services at very reasonable prices. By making it cost-effective to outsource protein production, GenScript has made it possible for my lab to focus on our own area of expertise and get more research done. The detailed planning, updates, and reports that GenScript provides all of the quality control that one could ask for. I strongly recommend GenScript's protein production service."
— Dr. Barry Bradford, Kansas State University, Department of Animal Sciences & Industry
Watch the video: Πόση πρωτεΐνη χρειάζομαι την ημέρα; οδηγός διατροφής (June 2022).