Protein Sciences (Biomolecular)

Protein Purification is a popular protein science service offered by Blue Sky Biotech.

Other protein science services included but are not limited to the following:

• FPLC: IEC, IMA, SEC, AC, Buffer-exchange, etc.
• Microsome preparation
• Tissue/organ extraction and purification
• Antibody purification
• Development of multi-step protocols
• Activity validation (where applicable)
• QC supported by MASS Spec service
• QC by Western Blot and SDS PAGE

Purified recombinant protein is indispensable for drug discovery.  In the early stages of drug discovery, recombinant protein is used for target validation, enzyme characterization, high-throughput screening (HTS), and structural study.  In order to understand a disease target, pharmaceutical scientists must study its interaction with substrates.  For this purpose, researchers need active protein free of impurities which could interfere with their study of its mechanism.  Purity and activity of the recombinant protein are essential:  a pure, active enzyme gives a clean signal and reproducible results.

Blue Sky employees have worked in the Protein Science department of a major pharmaceutical company.  This group and others like it serve almost as "core facilities" in that they provide made-to-order proteins for enzyme characterization, structural study, and HTS.  Our experience in this environment gives us a unique understanding of the requirements for quality, purity, and activity of recombinant proteins. Customers of Blue Sky may rest assured that all purified proteins provided by this firm will meet the most exacting standards.

Protein expression, purification, and characterization are very precise, difficult disciplines.  We at Blue Sky have the expertise to generate the high-quality protein reagents which you need.  

Our most common protein services include the following:

• Generation of expression strains in E. coli and yeast ( S. cerevisiae, P. pastoris, S. pombe) systems; expression of recombinant protein in E. coli and yeast systems
• Generation of recombinant baculoviruses and protein expression in insect cells with he baculovirus system
• Protein purification by affinity, ion-exchange, and size-exclusion chromatography
• Protein analysis by gel electrophoresis and mass spectrometry

Other, less-common services include the following:

• Expression of recombinant protein in mammalian cells by plasmid- and retrovirus-based systems
• Protein purification by reverse-phase HPLC and other methods of chromatography
• Protein analysis by 2-D electrophoresis, N-terminal sequencing, and other methods

Demystifying Baculoviral Expression Replicability

Insect cells are the preferred module for high-level recombinant protein expression of many disease-relevant protein families. Several popular kit systems exist in the protein expression marketplace, however all common systems utilize baculovirus as a vector for host infection and protein production.

The most challenging element of baculoviral-mediated (baculo) protein expression is low batch-to-batch (lab-to-lab) replicability. Blue Sky has developed a multi-component system which employs Infection Kinetics Monitoring (IKM) technology. This system compiles a suite of real-time cell population measurements, read during the course of viral infection.

In tandem with standard scouting measures (such as Western Blot), IKM substantially decreases the guesswork associated with scale-up baculo expression. This system also obviates the need for laborious baculoviral titration. IKM makes baculo expression more predictable.

Molecular Biology Services

• Gene synthesis
• Sub cloning and plasmid construction
• Vector construction and modification
• Construct generation
• Site-directed mutagenesis
• Genetic (cDNA) cloning
• In vitro transcription
• Protein expression scouting
• Plasmid preparation

Molecular biology drives modern drug discovery.  Drug screening, target validation, and structural studies all require recombinant DNA constructs and proteins.

Though molecular biology techniques are extremely common and widely-used, they can also be very tiresome, tricky, time-consuming, and frustrating.  In addition, equipment for molecular biology is extremely expensive.  In many cases, it is simply more economical to outsource this work than to perform it in-house.

Blue Sky will help you to fulfill your needs for recombinant DNA constructs and for other molecular biology services.  From the initial cloning of your gene of interest to the development of multiple constructs and site-directed mutagenesis, we will provide a comprehensive suite of services to move your project along quickly and smoothly.

Our most common services include the following:

• Genetic (cDNA) cloning
• Subcloning and plasmid construction
• Vector construction and modification
• Construct generation
• Site-directed mutagenesis
• Generation of recombinant baculoviruses
• Transient and stable transfection of mammalian cells

Tissue Culture & Fermentation

• Scale-up of E. coli in 10-L/55-L bioreactors
• Scale-up of yeasts in multiple 10-L bioreactors
• Scale-up of insect-cell and mammalian expression in bioreactors
• Optimization of expression in bioreactors
• Process development for fermentation
• Perfusion and microcarrier culture
• Batch, fed-batch, and continuous culture
• Intracellular and secreted products
• Stable Cell line production
• Membrane preparation

Life for the protein scientist would be very easy if every protein could be purified in hundred-milligram quantities from small-scale flask cultures of E. coli.Unfortunately, many proteins require expression in animal-cell systems and culture scale-up.

Blue Sky scientists will take care of the frustrating, laborious work of tissue culture- our clients can save their time for true scientific investigation instead of spending hours ensuring that cultures stay healthy and contamination-free. We have a great deal of experience with insect-cell lines, and in our past work we have grown dozens of different mammalian cell lines.

In similar fashion, Blue Sky will save its clients a great deal of time with bioprocess scale-up services. Using bioreactors, Blue Sky scientists will quickly deliver large quantities of cell paste or conditioned medium.

Our most common services include the following:

• Insect-cell culture (Sf9, Sf21, HighFive, Drosophila Schneider S2, and other lines)
• Production and amplification of recombinant baculoviruses in insect cells
• Process development in baculovirus expression
• Culture, transient and stable transfection, and subcloning of common mammalian cell lines (293, CHO, COS, 3T3, HeLa, and others.
• Scale-up of bacterial and yeast cultures in laboratory-scale (up to 10-L) bioreactors
• Scale-up of insect-cell and mammalian cultures in laboratory-scale (up to 10-L) bioreactors
• Monoclonal antibody production from established hybridoma lines

Less-common services:

• Culture, transient and stable transfection, and subcloning of less-common mammalian cell lines
• Perfusion culture of animal cells

Adult Stem Cells Show Wider Potential Than Previously Thought

Chemists are developing new insights and techniques in an effort to expand the therapeutic potential of stem cells, which includes possible treatments for Parkinson's disease, diabetes, spinal cord injury and other devastating conditions.

Embryonic stem cells are the most versatile stem cells, capable of being transformed into any other cell type, depending on their desired therapeutic use.

Now, researchers at Northwestern University have found new evidence that hematopoietic stem cells, a type of adult stem cell derived from the bone marrow that gives rise to blood cells, are capable of undergoing more diverse transformations than previously thought and could be transformed into a wide variety of tissue types, not just blood cells.

In recent laboratory tests, human megakaryocytes (bone marrow cells that produce blood platelets that are responsible for blood clotting) derived from adult hematopoietic stem cells were, for the first time, reprogrammed into neutrophil-like cells similar to the white blood cells that are responsible for fighting infections, according to study leader E. Terry Papoutsakis.

Insights from this study could help guide similar adult stem cell transformations in other cell types in the future, he says.