How to Increase Protein Production Capability?

With the common effort of the staff scientists and researchers, Creative Biolabs released its Single Protein Production (SPP) ^TM technology into the protein production market. This service is developed mainly for the R&D of high-throughput protein production.

In the SPP system, live E. coli cells are converted into a bioreactor producing only a single protein of interest in a high yield. A yield of 20–30% of total cellular protein can be obtained with our technology, which overwhelms all protein production methods known so far.

Its technology involves the introduction of an mRNA endoribonuclease or interferase in E. coli cells to disrupt the endogenous protein production. However, the cells retain full metabolic activity for RNA and protein synthesis. Therefore, when the mRNA for a protein of interest is engineered to be devoid of the mRNA endoribonuclease recognizing sequence without altering the amino acid sequence of the protein, the cells start to produce any single protein of your choice.

With the evolution of this technology, Creative Biolabs is now capable of producing any proteins with the SPP system. With the technology, protein yields can be kept without being affected even if the culture is condensed up to 40-folds, reducing the cost of protein production by up to 97.5%. The technology provides isotope-labeled proteins at a very high signal-to-noise ratio. More than 90% of the isotope can be incorporated into the target protein in the SPP system. Furthermore, a refinement of this technology has eliminated lengthy purification steps in the production of membrane proteins suitable for structural studies. 

Learn more about Single Protein Production (SPP) ^TM technology of Creative Biolabs.

About Creative Biolabs

Creative Biolabs, a US biotech company established in 2005, is focused on the research and development of antibody engineering and protein production. Learn more about Creative Biolabs. 

The Future of Sequencing Technology in Clinical Application

As science develops, traditional Sanger sequencing has failed to meet the requirements of low cost, high throughput and fast in speed. It is under this circumstance that the next-generation sequencing technology (second-generation sequencing) appears. As a comparatively new industry, the next-generation sequencing technology can be applied for clinical genetic testing, health industry, industrial and agricultural use of gene-oriented study as well as scientific research and development.

Recent years, with the discovery and promotion of second-generation sequencing technology, gene sequencing speeds up greatly while achieving a substantial decline in costs, making large-scale application of genome sequencing possible. Now, the cost of personal whole genome sequencing is about 5,000$, and is expected to decreased to less than $ 1,000 in the next few years.

The rapid development of sequencing technology will promote the massive accumulation of DNA sequencing data, accompanied by the accumulation of the corresponding individual signs, diseases and other data at the same time. When enough data is accumulated, how to understand these data will be critical. On the micro level, generations of molecular biologists' studying the effects of apparent biological traits genes exert on utilizing technologies (such as gene knockout) has made breakthroughs in many crutial aspects. On the macro level, statistics and other data analysis techniques are introduced to study the relationship between gene sequences and biological phenotype. The accumulation of basic scientific research gradually brings breakthroughs in clinical applications.

There are now two types of clinical applications mainly, one aims at disease screening of ordinary people. It detects people’s risks of getting cancer in the future by measuring the known genes associated with a disease loci. The other aims at the diagnosis cancer and other deadly diseases. It finds in a series of drugs or plans the most effective one for certain patients by testing the loci of certain genes.

Data from BBC research shows that total global gene sequencing market increased from $ 7.941million in 2007 to $ 4.5 billion in 2013, and is predicted to reach $ 11.7 billion in 2018 with the CAGR up to 21.2%.

Currently, the market of next-generation sequencing platform is mainly taken by several major manufacturers, including the Illumina, Ion Torrent / Life Technologies (was the acquisition of Thermo Fisher in 2014), 454 Life Sciences / Roche and other small ones like Creative Biolabs and CD Genomics.

Current Situations of Genome Sequencing for New Born Babies

Since last century, newborn babies have gotten a heel-prick test in which their blood is screened for dozens of congenital diseases. Routine newborn baby screening has basically eliminated the risk of death or irreversible brain damage that some of these disorders can pose if they are not identified right away. Even though, it has brought about great controversies in this field.

Last December, Mercy Children’s Hospital of Kansa released its results of genome sequencing for sick new born babies on Science Translational Medicine. In this research, they practiced whole genome sequencing or exome sequencing for children with severe neurological developmental disorders from 100 families, among which there are families that have been seeking for diagnostic methods for their children for years. According to the data, 45% of the families have experienced genome sequencing. Moreover, 73% of the babies with congenital disease have accepted this brand new testing approach.

Doctor Stephen F. Kingsmore, leader of this project hopes that the whole genome sequencing project could be carried on to around 14% of the newborns observed in ICU among the total 4 million per year.

Researchers from the USA are also investigating families’ attitudes towards genome sequencing. Last December, they made a research on parents of 514 healthy babies born within the past 48 hours in Brigham and Women's Hospital. This research was aimed to get a general knowledge of how much do these parents know about their babies’ genome information, risks of getting genetic diseases and the meaning of genome information. While being asked about whether they would be willing to attend the project of whole genome sequencing for newborn babies, results showed that 83% said yes and were happy to be in this project.

Moreover, Donald Chaplin of Acton, the farther a 17 months old baby, was also interested in it. He himself is a pharmacist, and he worried that the gene data might have the risk of been misused. Even though, he still wants to learn more about the gene information of his son. Jamaican engineer Nicholas Catella, however, said that he wouldn’t care much about the result unless it shows that his babies have great risks of getting severe diseases. Mr. Catella has two children, one is three old and the other 16months. He knows that gene sequencing is able to reveal the risks of getting chronic diseases like Alzheimer's, but we do have no good invention methods at present.

We hope, one day when this new testing skill develops greatly, that the gene sequencing data since the birth of babies can accompany throughout all their life, and that gene sequencing can lead the growing process of newborn babies. 

Bivalent and Bispecific Single Chain Antibody in Solving the Problems of scFv

Antibody is not a stranger to everyone, even a new born baby. It is the black hole on the biological world, as there is always something new that attracts biologists and researchers.

Since the first success achieved in single chain antibody fragment（scFv）research by Bird and Huston in the 1988, tremendous achievements have been made in the research and development of single chain antibody technology.

However, in terms of application, there are still problems remain unsettled. The scFv fragments derived from phage display antibody libraries usually have short half-life and less affinity. However, multivalency of antibody molecules turns out to be a desirable property in many in vitro and in vivo applications. After years of experience and great efforts, scientists and researchers from Creative Biolabs have carried out a series of approaches for bivalent and bispecific scFv and Fab construction, which may greatly contribute to the current situation. (Bivalent and Bispecific scFv/Fab)

According to its newest data, there are three approaches of generating genetically engineered and dimerized scFv antibody fragments, miniantibody, diabody and Tandem scFv.

Figure 1. Schematic diagram of bispecific antibody

Miniantibody

Bivalent or bispecific (scFv) 2, the so-called miniantibody, is produced by the combination of two scFv molecules with two modified dimerization domains. Leucine zippers are utilized to mediate dimerization of scFv in a miniantibody form. It constructed dimerization cassettes which allow the conversion of scFv antibodies from all its phage display libraries to bivalent or bispecific antibodies. During this procedure, either Fos or Jun leucine zippers are fused to scFv proteins. Two cysteine residues were engineered in the Fos and Jun zipper domains to produce disulfide-stabilized homodimers, which usually leads to efficient production of stable, secreted homodimers that are able to retain their specificity as assessed in a number of assays.

Diabody

Diabody is a non-covalent dimer of single-chain scFv, fragments that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker. 14–15 amino acid residues’ common linkers are long enough to span the distance between the N- termini and C-termini of the variable domains in a scFv. However, utilizing linkers of 3–12 amino acid residues in length can lead to the formation of diabody.

When two ScFvs linked with the short linkers are expressed in the same cells, dual-functional antigen-binding sites will be formed through crossover pairing of the variable light-chains and heavy-chains. The bi-specific diabody, which is constructed with heterogeneous scFvs, is also an important and commonly used form of recombinant bi-specific antibody. A distinct feature of diabody is that it has a rigid structure and can be expressed at high yields in bacteria. See figure 2.
It is worth mentioning here that bispecific T cell engager (BiTE) is a unique form of tandem scFv. In a BiTE molecule, one of the scFvs binds to T cells via CD3 receptor and the other to a tumor cell via a tumor specific molecule. As this procedure brings together the T cell and cancer cell, it has great possibilities in cancer therapy. (BiTE). See figure 2.

Tandem scFv

Tandem scFv (taFv) is produced by connecting two scFv molecules with a short linker. This form of scFv has a very flexible structure and is comparatively easy to be generated. Both bacterial expression and refolding and eukaryotic expression are able to produce tandem scFvs. With years of experiences, Creative Biolabs has successfully constructed over 100 tandem scFvs.

Figure 2. Schematic diagram of Diabody and Tandem scFv

About Creative Biolabs

Creative Biolabs is a professional biotech service provider. Since been established in the year of 2005, it has been focused on the research and development of single chain antibody technology. Learn more about Creative Biolabs.

Single Domain Antibody Made It Possible for Curing Diseases by Taking Antibody Drugs

The first single domain antibody being engineered from the heavy-chain antibodies found in camelids has greatly stimulated the development of antibody market.

The Rapid Development of Antibody Drugs

Except brain, the most sophisticated part in a human body is the immune system. In a sense, we are living in a world surrounded by various bacteria. They never cease disturbing us by taking over our living surroundings as their ideal place for breeding. Under this circumstance, antibody is the only one that can take the responsibility of an inner protector. Each antibody model has its own mission. When foreign objects, including microorganism, allergen and toxin, are trying to invade the immune system, antibodies will send out unique chemical signals on receiving which our immune system starts working.

Even with such a complicated self-defense system, we still suffer a lot from disease. While confronting with cancer or respiratory virus infection, the immune system always delays the response. Sometimes it overacts in an asthma attack or when rejection occurs in organ transplantation. It may even mistakenly attack normal cells and leads to rheumatic arthritis and other autoimmune diseases.

For years, pharmaceutical researchers have been trying to create an engineered antibody that can overcome or relieve these immune system diseases. With the efforts of a large group of research team, Creative Biolabs, a US biotech company, successfully engineered single domain antibody which is also called single domain antibody from the heavy-chain antibodies found in camelids. Creative Biolabs owns its success to three factors, the rapid development of therapeutic antibody, the disturbing problems related to antibody drugs and the unique understanding of camelids concerned biological knowledge by its scientists.

Why It Attracts So Much Attention?

Compared with normal antibodies, this distinct antibody, with only 10% of their molecular mass, is more flexible in terms of chemical property. Thanks to its tiny size and non-hydrophobic property, it performs better in heat as well as acidic and alkali resistances. In the experiment on mice, their being able to remain viable and stable while passing through the mice’s digestive system has further proved the possibility of curing diseases by taking antibody drugs.

Based on all these advantages, Creative Biolabs widened its services to meet more requirements from the market, including the construction of immunized antibody libraries of this specialty and synthetic camelised human single domain antibody libraries with the usage of llama and camel and DNA synthesis respectively. Bio-panning of single domain antibodies libraries and large scale production of recombinant single domain antibodies are also the fields of its expansion project.

About Creative Biolabs

Creative Biolabs is a professional service provider in the field of single domain antibody production and single domain antibody libraries. It is specialized in generating single domain antibodies against any targets. 

No End in Bacteria Research and Development

Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. We can find bacteria in soil, water, acidic hot springs, radioactive waste, and the deep portions of Earth's crust. Moreover, they may even flourish in manned spacecraft. Among all, however, the most common one is to be found in plants and animals that bacteria are living in symbiotic and parasitic relationships with.

Why Scientists Put Much Strength in Bacteria Research and Development?

Bacteria exist among all our surroundings, thus research into them has become an essential task. Scientists never stop their steps in moving further in this field from its fist discovery in the 16^th century by Antony van Leeuwemhoek, the farther modern microbiology. As the development of biotechnology, scientists now can use more advanced technique to push forward this research, bacterial display.

Why We Need Bacterial Display?

Bacterial display or bacterial surface display is a commonly used protein engineering technique, especially in terms of in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures. This protein engineering technique allows us to put together the function of a protein and the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinity ligands which are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification of enzyme substrates and finding the affinity of a ligand for its target protein.

Hi-affinity^TM Bacterial Display Technology for Creating Therapeutic Antibodies

Hi-affinity^TM, offered by Creative Biolabs, is a unique branch of bacterial display technology based on a proprietary synthetic bacterial display human antibody library and a unique selection process natural to E. coli. This bacterial surface display technology enables the rapid isolation of target-specific antibodies without the labor intensive screening common to other recombinant and non-recombinant antibody production methods, resulting in unique single-chain variable fragment (scFV) antibodies that have both high specificity and extremely high affinity [Kd up to10-12] for the target antigen.

Creative Biolabs created the Hi-affinity^TM human scFv antibody library by combining a highly diverse collection of synthetically-constructed randomized CDR sequences that are further diversified by random lengths using a unique proprietary technique. The large library has been specifically optimized to eliminate unwanted stop codons and aggregation-prone sequences. scFv molecules are first expressed in E. coli cytoplasm and then translocated and anchored into the bacterial plasma membrane. In the end, binder panels are selected by FACS or panning. Furthermore, multiple rounds of affinity maturation during library screening are incorporated through error prone PCR mutagenesis either directed at CDR or flanking sequences and selection by varying antigen dose.

Hi-affinity^TM platform of Creative Biolabs utilizes a unique antibody selection process that relies on the natural twin-arginine translocation (Tat) system in E. coli. The featured benefit of Hi-affinity^TM is its capacity of generating human antibodies with exceptionally high affinity.

From the starting point of knowing the existence of bacterial to research into them and put them into biotech development by now, it is easy to predict that there is more for human to explore.