Protein FAQ

A CAR (Chimeric Antigen Receptor) is a genetically engineered receptor that combines the antigen-binding ability of an antibody with T-cell activation capabilities. The CAR structure comprises four main components:

The CAR positive rate is a critical metric for evaluating CAR-T product potency, directly influencing target recognition and cytotoxic efficacy. Monitoring CAR expression in peripheral blood or tissues post-infusion provides insights into in vivo expansion and persistence, informing treatment response, durability, and potential relapse risk. Dynamic CAR expression monitoring also helps assess immunogenicity and safety. Thus, a sensitive, reliable, and universal CAR detection system is vital for developing and evaluating CAR-T cell therapies. Currently, approved CAR-T products target CD19 or BCMA, making flow cytometry with recombinant BCMA or CD19 protein the standard detection method. Although highly specific, this approach is limited to single targets and is unsuitable for CAR-T screening and preclinical development. Several CAR detection methods have been developed, including Protein L (which binds immunoglobulins) or Fc-binding polyclonal antibodies. Protein L binds only to kappa light chains and fails to detect CARs containing lambda chains. Polyclonal antibodies may cross-react with patient IgG, causing false positives. Anti-idiotype antibodies offer high specificity and sensitivity by targeting unique idiotopes in the CAR's antigen-binding region, but they are challenging to develop and produce.

Stupp and his team posited that "dancing molecules" might encourage the stubborn tissue to regenerate. Previously invented in Stupp's laboratory, dancing molecules are assemblies that form synthetic nanofibers comprising tens to hundreds of thousands of molecules with potent signals for cells. By tuning their collective motions through their chemical structure, Stupp discovered the moving molecules could rapidly find and properly engage with cellular receptors, which also are in constant motion and extremely crowded on cell membranes. Once inside the body, the nanofibers mimic the extracellular matrix of the surrounding tissue. By matching the matrix's structure, mimicking the motion of biological molecules and incorporating bioactive signals for the receptors, the synthetic materials are able to communicate with cells. "Cellular receptors constantly move around," Stupp said. "By making our molecules move, 'dance' or even leap temporarily out of these structures, known as supramolecular polymers, they are able to connect more effectively with receptors."

Stupp's team is currently testing these systems in animal studies and adding additional signals to create highly bioactive therapies. "With the success of the study in human cartilage cells, we predict that cartilage regeneration will be greatly enhanced when used in highly translational pre-clinical models," Stupp said. "It should develop into a novel bioactive material for regeneration of cartilage tissue in joints." Stupp's lab is also testing the ability of dancing molecules to regenerate bone - and already has promising early results, which likely will be published later this year. Simultaneously, he is testing the molecules in human organoids to accelerate the process of discovering and optimizing therapeutic materials. Stupp's team also continues to build its case to the Food and Drug Administration, aiming to gain approval for clinical trials to test the therapy for spinal cord repair. "We are beginning to see the tremendous breadth of conditions that this fundamental discovery on 'dancing molecules' could apply to," Stupp said. "Controlling supramolecular motion through chemical design appears to be a powerful tool to increase efficacy for a range of regenerative therapies." The study, "Supramolecular motion enables chondrogenic bioactivity of a cyclic peptide mimetic of transforming growth factor-β1," was supported by a gift from Mike and Mary Sue Shannon to Northwestern University for research on musculoskeletal regeneration at the Center for Regenerative Nanomedicine of the Simpson Querrey Institute for BioNanotechnology. Yuan, S. C., et al. (2024). Supramolecular Motion Enables Chondrogenic Bioactivity of a Cyclic Peptide Mimetic of Transforming Growth Factor-β1. Journal of the American Chemical Society. doi.org/10.1021/jacs.4c05170

Affimers can be combined with traditional biological tests to increase our understanding of proteins. For example, affimers have been used in affinity assays, among others. They have also been used in biosensors and in cell cultures to manipulate cell signaling. Affimers have been successfully applied both in vitro and in vivo. Some of the applications that have been tested using affimers include cell imaging, super resolution microscopy, protein function modulation, magnetic nanoparticle formation, and MRI reagent development. Affimers with enhanced specificity for tubulin have been used in super resolution microscopy with high success, and can target regions of the tubulin structure that antibodies are typically restricted from. It is likely that more applications will be discovered in the future as the use of affimers becomes more widespread. For example, thanks to growing libraries of affimers, it has been possible to create affinity microarrays to discover biomarkers and potential pharmaceuticals. By finding biomarkers, affimers have been applied to cancer studies, where they have been used to target a protein in tumor vessel formation called the VEGF receptor.

Affimers stem from the drive to generate alternative methods of studying protein binding and function, which can complement information provided by antibodies. As a type of affinity reagent, affimers are biophysically stable scaffolds of proteins that can be reused. Affimers can be made from two scaffolds: Adhiron scaffolds and human stefin A scaffolds. The stefin A scaffold is engineered from human stefin A protein, whereas Adhiron is synthetic. The Adhiron synthetic protein was originally made from the sequence of cystatin, and is very thermally stable. The Adhiron and stefin A scaffolds are structurally related. To select an appropriate affimer, screening is done to find suitable reagents for the target protein. The chosen coding regions are then cloned into an expression vector in Escherichia coli, and the recombinant protein was purified over the course of a week. This allows for generation of high quality proteins for diverse applications.

Affimers are small in size, stable at different temperature ranges, relatively easy to generate, and offer appealing opportunities to complement the shortcomings of antibodies. Their small size is thought to be one of the attributes making affimers more sensitive than antibodies when used in histochemical staining, as they can penetrate tissues better. Affimers are both commercially available and can be synthesized by individual laboratories using publicly available methods, thereby ensuring it is accessible to the entire scientific community. One key advantage of affimers, as opposed to antibodies or nanobodies, is the speed at which they can be screened and developed. The screening process for affimers involves three iterations of phage display and ELISA assays. This can be carried out in 12 days, which is significantly less than the time needed for antibody and nanobody animal inoculation times. Furthermore, the screening process by which binding reagents are identified is more efficient because the target proteins are screened in a folded state.

The study by Williams was looking for variations in hemoglobin, the oxygen-binding protein found in red blood cells. There are disorders which lead to abnormalities in hemoglobin, which can cause serious illness or even death. Identifying these variants would lead to a better prognosis for people with such abnormalities, and the study found a variant by having the ability to differentiate between leucine and isoleucine. Methylmalonic acidemia (MMA) and propionic acidemia (PA) are metabolic disorders resulting from an inability to break down isoleucine, valine, methionine, and threonine (which are also amino acids), odd-chain fatty acids, as well as cholesterol. These are rare diseases which are known as “inborn errors of metabolism” (IEM). The restriction of certain foods and the use of medical foods can form part of the treatment for IEM disorders. Leucine is added to a lot of these medical foods, and is hence selected for patients with MMA and PA. As patients with MMA and PA cannot break down isoleucine and valine, foods containing these amino acids should be avoided. However, it was previously reported that avoiding such amino acids can lead to an imbalance in the amounts of branched chain amino acids in the blood. More specifically, the ratio of leucine to isoleucine/valine was found to be increased. This could have dire consequences, potentially disrupting the synthesis of neurotransmitters due to impaired amino acid transport into the brain. In this case, it would be pivotal to distinguish between leucine and isoleucine, as the balance between these two amino acids needs to be restored to avoid potential damage.

Leucine and isoleucine are transported into organs (including the brain) by the use of L1-neutral amino acid transporter, or LAT1. As other amino acids are also transported by LAT1, leucine and isoleucine must compete for a binding site. Amino acids imported into the brain can be used to synthesize neurotransmitters, therefore changes in amino acid concentrations may have an effect on the neurotransmitters that are made. Leucine has additional roles; it may inhibit breakdown of muscle, causes the secretion of insulin from pancreatic β-cells, as well as modulate food intake regulation through its action on the central nervous system.

The alpha-synuclein (α-syn) protein is found in various locations around the body, such as the heart, the skeletal muscles, and the gut. However, it is most abundant in the human brain. Here, the protein is found concentrated in the synapses of the neurons and is related to the function of transmission of chemicals across the synaptic cleft. This process of neurotransmission is an essential process that takes place constantly in the brain and is implicated in functions from learning and memory to speech and movement. De-regulation of neurotransmission has been linked with a myriad of psychological and neuropathological diseases. While the role of the alpha-synuclein is still not completely understood, research has given some insights into what, potentially, are its main functions. Evidence has revealed that alpha-synuclein is involved in the regulation of the transportation of dopamine from one neuron to the next. Dopamine is a neurochemical that is responsible for controlling both movements and emotional responses, it is known to have a critical role in Parkinson’s disease (PD), with the loss of cells that produce the substance resulting in a lack of movement control, leading to the characteristic movement symptoms of the disease.

As mentioned before, differentiating leucine and isoleucine could be cumbersome due to the fact that they are isomers. To overcome this, a study by Sowell and coauthors combined liquid chromatography and mass spectrometry on the example of Maple Syrup Urine Disease (MSUD). MSUD represents a condition where an accumulation of branched-chain amino acids (mostly leucine) is observed in the blood, stemming from a defect in step two of the breakdown pathway. One way of diagnosing MSDU is to analyze blood samples to see how much branched chain amino acids are present. Using liquid chromatography first, the authors showed that it was possible to differentiate leucine and isoleucine. A similar method was used by Williams and coauthors to also distinguish leucine and isoleucine; a mass spectrometry-based approach using hot electron capture dissociation was employed in this example, with a Fourier transform ion cyclotron resonance mass spectrometer. Here, there is excess energy which leads to extensive fragmentation of the protein (or peptide) being analyzed.

Food-derived proteins are broken down during digestion releasing amino acids which are then used by the body to create proteins required by bodily organs, tissues, and cells. The body can code for 20 different types of amino acids. However, some amino acids are considered to be essential whereby they cannot be synthesized in large enough quantities by the body. Therefore, they must be consumed as part of a balanced diet. Non-essential amino acids are produced by the body. There are nine types of essential amino acids: phenylalanine, histidine, threonine, isoleucine, leucine, tryptophan, lysine, valine, and methionine. Of the nine essential amino acids, leucine, valine, and isoleucine are referred to as branched-chain amino acids. Generally, all three BCAAs act as a substrate to produce protein and energy. Leucine plays an additional role as an intercellular signaling regulator for the mechanisms of protein production. Typically, BCAAs are found in foods rich in proteins such as eggs, fish, red meat, dairy and chicken as well as soy proteins, nuts, and lentils. Research has found that as the production of protein is continuous, there is a need to replace lost protein. This need for continued protein intake has increased the popularity of BCAA supplements. For example, the inclusion of BCAA supplements into the diets of bodybuilders, athletes, and gym-goers has increased due to their proposed muscle-building benefits and ability to enhance performance.

There is mixed evidence regarding the effectiveness of BCAA supplements to increase muscle mass in its users. Research investigating such effects between those who took BCAA supplements and a placebo found that those who took the supplement reported increase energy metabolism and reduced levels of substances attributed to muscle damage. However, other researchers proposed there is a lack of consensus regarding the effects of BCAAs as a nutritional supplement. Despite disagreement in the scientific literature, it is indicated that potential overconsumption of BCAAs may pose health risks. However, more research is needed to further investigate the impact of both typical and excessive consumption.

Reporting in the October 13 issue of Nature, a team from Denmark-based biotech company Novozymes, and researchers from Georgetown University Medical Center and the David Geffen School of Medicine at UCLA, say they have isolated "plectasin," the first defensin ever found in fungi. The research was performed at Novozymes laboratories in Denmark. Defensins are peptides, miniature protein molecules that are produced by a wide range of animals to protect themselves against infection. Humans have defensins in their white blood cells and in their skin, for example, but it is believed that this new fungal defensin, plectasin, is more potent and targets certain bacteria more specifically. Indeed, when plectasin was tested in the laboratory and in animals, it proved to be highly effective against the bacteria Streptococcus pneumoniae, and Streptococcus pyogenes, including strains that are now resistant to conventional antibiotics. These bacteria are responsible for such diseases as meningitis, community-acquired pneumonia, strep throat, life-threatening sepsis, and flesh destroying skin infections. The discovery of plectasin has implications for the development of defensins as a treatment against many common, and deadly, infections, and may initiate a new era of antibiotic discovery and development, said study co-author Michael Zasloff, M.D., Ph.D., Professor in the Departments of Surgery and Pediatrics at Georgetown University Medical Center. Zasloff says that the field of antibiotic development has not changed much since 1929 when Alexander Fleming realized that the fungal "bread mold" Penicillium, which had landed by chance in a Petri dish produced a substance that eliminated colonies of staphylococcal bacteria. "Most antibiotics used by humans are produced by fungi and certain soil bacteria," he said. "Using our existing tools of discovery, we have failed to uncover any new classes of antibiotics from these sources over the past decade. However, by utilizing a new genetic approach that allowed the team to discover plectasin, we now know that a whole class of antibiotics has been overlooked." "This finding (plectasin), and the existence of about 200,000 additional species of fungi, opens up a vast universe to explore for novel peptide antibiotics," said co-author Robert Lehrer, M.D., Distinguished Professor of Medicine at the David Geffen School of Medicine at UCLA. Plectasin, if proven safe and effective in humans, could be on the market by 2012, said Lehrer. Zasloff and Lehrer are known internationally as experts in antimicrobial peptides - the class of antibiotics that plectasin falls within - and in this study they collaborated with Novozymes, a Danish biotech company that led the research. Zasloff and Lehrer are the only two scientists from U.S. universities on the team of 20 researchers who co-authored the research paper. All life forms have to defend themselves against microbial invaders - bacteria, fungi, viruses - and to do this, they produce antimicrobial defensin peptides. In humans, defensins are made by specific white blood cells and immune cells that later engulf foreign invaders, and by the skin and mucous membranes, in order to kill microbes before they invade protective barriers. Researchers believe that fungi have a similar system of defense, especially since these plant-like organisms live off rotting matter, said Zasloff. "They must compete with other organisms, like bacteria and viruses, which also want to consume the same meal. In addition, they need to defend themselves from being eaten by the microbes which surround them." But he said no one had been able to find defensins in fungi using traditional research techniques, which involved growing fungi in liquid cultures and then testing the culture to see if it contained any antibiotic molecule. The research team instead used the latest genetic science to search for the defensins they thought fungi must have. Selecting the Pseudoplectania nigrella species of fungus may have been serendipitous, Lehrer said, but the Novozymes team used state-of-the-art biotechnology to intercept ,and interpret its genetic messages and exhibited tremendous skill in producing plectasin efficiently, economically, and in large amounts." "I started working on antimicrobial peptides over three decades ago, said Lehrer, and my laboratory first described human defensins in 1985. So, the discovery of plectasin makes me feel like a grandfather." Further examination revealed that this defensin, plectasin, resembles defensins found in spiders, scorpions, dragonflies and mussels - thus suggesting that the defensins found in insects, molluscs and fungi arose from a common ancestral gene, the researchers say. Based on this information, the scientists now believe that defensins appeared in living things more than a billion years ago. The investigators then turned to the National Center for Antimicrobials and Infection Control, the Danish equivalent of the U.S. Centers for Disease Control, to test plectasin in the laboratory for antimicrobial activity against a broad spectrum of bacteria. It showed potent activity against several species of Gram-positive bacteria, and was especially active against S. pneumoniae (the leading cause of pneumonia), including all known clinical strains and those that are now resistant to conventional antibiotics. "That is important because increasing bacterial resistance to conventional antibiotics threatens the future of many antibiotics in current use," Zasloff said. "In mouse studies, plectasin showed extremely low toxicity, and was as effective as vancomycin and penicillin in curing the animals of experimental peritonitis (inflammation of the lining of the abdominal cavity, which can be deadly) and pneumonia caused by S. pneumoniae, the researchers report. "Although the precise mechanism by which plectasin exerts its antimicrobial activity is still under investigation, it may work by a mechanism that is very different from traditional antibiotics, Zasloff said. "As a group, defensins exhibit activity against many types of bacteria, fungi, protozoa, and even viruses. It is entirely possible that fungal defensins will be discovered that could be developed against all of these human pathogens," Zasloff added.

Excess carbohydrates in the diet are converted into triglycerides, which involves the synthesis of fatty acids from acetyl-CoA in a process known as lipogenesis, and takes place in the endoplasmic reticulum. In animals and fungi, a single multi-functional protein handles most of these processes, while bacteria utilize multiple separate enzymes. Some types of unsaturated fatty acids cannot be synthesized in mammalian cells, and so must be consumed as part of the diet, such as omega-3. Acetyl-CoA is also involved in the mevalonate pathway, responsible for producing a wide range of isoprenoids, which include important lipids such as cholesterol and steroid hormones.

Lipids are mainly composed of hydrocarbons in their most reduced form, making them an excellent form of energy storage, as when metabolized the hydrocarbons oxidize to release large amounts of energy. The type of lipid found in fat cells for this purpose is a triglyceride, an ester created from glycerol and three fatty acids.

For the average adult, the Recommended Dietary Allowance (RDA) for protein or the amount you need to meet your basic nutritional requirements and not get sick, is 0.8 grams of protein per kilogram of body weight, or 0.36 grams per pound. For a person who weighs 75 kg (165 pounds), that comes to 60 grams of protein per day. To determine your target daily protein intake, you can multiply your weight in pounds by 0.36, or use this online calculator. It's important to keep in mind that your daily protein need is not a fixed number. For example, your protein needs will fluctuate depending on your level of physical activity, and whether you are pregnant or breastfeeding.

The idea of an upper limit of protein intake is controversial. Some experts believe the recommended amounts are too low. Others believe we are overconsuming protein. In general, the average person (not a body builder or an elite athlete) does not need high levels of protein and should not exceed 2 grams per kilogram; or about 125 grams per day for a 140-pound person. A main concern with taking in too much protein is an overall dietary imbalance. Consuming excessive protein may lead to inadequate intake of carbohydrates and fats. It's important to maintain a well-rounded diet that includes a balance of all three macronutrients for overall health. In addition, if you increase your protein intake without increasing physical activity, you may be taking in too many calories without expending enough, which could result in weight gain. There is also some evidence that eating a relatively high-protein diet can be problematic for individuals with pre-existing kidney conditions. A diet high in animal protein may pose a higher risk of kidney stones.

We can get protein from plant or animal sources. Protein in our diet can come from meat, dairy products, nuts, some vegetables, and certain grains and beans.

In order to get enough dietary protein in the most healthful way, nutritional guidelines have shifted away from specific amounts of daily protein, and toward the importance of eating healthier, protein-rich foods. It's important to think about the protein "package" when making protein choices because food containing protein also includes fats, carbohydrates, vitamins, minerals, sugar, sodium, additives, and other components. While meat provides high-quality protein, some meats also provide unhealthy amounts of saturated fats and sodium. If you eat meat, it's important to choose leaner meats and poultry. According to the United States Department of Agriculture (USDA), meats that are high in saturated fat include: