Protein Sciences

RedSee™ Tag Platform

Fusion tags are engineered as chimeras with recombinant proteins for many uses such as affinity binding of known ligands (substrates) for the fusion protein. Accordingly recombinant protein fusions are easier to enrich and purify away from a constellation of other proteins based on this specific fusion/substrate binding (and release). Fusion tags have been around for 25 years, including the classic divalent cation:polyhistidine and glutathione:glutathione-S-transferase (GST) systems for expression and enrichment. It is impossible to apply polyhisitidine or GST fusion without time consuming analytical methods. RedSee™ is a native E. coli protein that binds available iron and emits red color (visible spectra). This protein was modified and constructed at Blue Sky using original publications dating back to the 1990s.

The ability to monitor expression visually (with the naked eye) is unknown prior to this technology. Fluorescent proteins have been in existence since the 1980s but require excitation and specific wavelengths. The ability for one to monitor the protein throughout a variety of stages—expression, binding, and enrichment/purification—makes it an attractive time-saving monitoring tool for the bench scientist. Evidence also suggests that the RedSee™ fusion also enhances solubility of some target proteins (proteins that may on their own be insoluble).

Puritin™ Tag and “Tandem Affinity Tag” (TAT)

Recombinant proteins are often fused to polypeptide tags with affinities for certain matrices that allow recombinant proteins to be purified using a defined process, obviating the need to develop a complex purification process, which can reduce the cost of protein production. Some examples of affinity tags include polyhistidine, Avi Tag, Glutathione-S-Transferase, Maltose-Binding Protein (MBP), and FLAG. However, single affinity purification steps often yield protein with sufficient purity for many applications, such as mass spectrometry, X-ray crystallography, or certain assays used in drug discovery. In recent years, the use of tandem affinity tags has grown in popularity because sequential purification utilizing two recombinant affinity tags has been shown to yield proteins with sufficient purity for most scientific applications.

Blue Sky Biotech has developed an affinity tag composed of a polypeptide derived from a mammalian protein that is covalently modified with biotin when co-expressed with another protein, biotin ligase. Biotin in turns binds with extremely high affinity to strepavidin matrices, thereby facilitating efficient capture of recombinant proteins. This tag is being utilized in combination with other affinity tags such as RedSee™ in our services offerings. The advantage of applying tandem affinity purification to Blue Sky’s production process is that it enables a common platform to be utilized for purification, thereby simplifying production, increasing efficiency, and improving the quality of finished products.

Polyhistidine Cap Technology

One of the most common peptide affinity tags in use today is polyhistidine because it is short and binds to a relatively inexpensive affinity resin. However, published data suggests that C-terminal polyhistidine tags can be cleaved by an endogenous enzyme(s), reducing or eliminating the utility of polyhistidine in applications requiring incorporation of affinity tags at the C-terminus of proteins.

Blue Sky Biotech hypothesized that degradation of polyhistidine might be due to the exposure of the tag and that an amino acid tail C-terminal to the tag might prevent its degradation. Addition of a single amino acid at the C-terminus of polyhistidine has now been shown to improve stability of C-terminal polyhistidine tags in all tests to date.

De-Tox™ Process and Endotoxin Reduction/Testing Platform

Endotoxins, or bacterial lipopolysaccharides (LPS), often contaminate recombinant protein preparations and are highly undesirable, especially when the protein preparations are to be used in immunological assays. Presence of endotoxin can cause false readings in cell based assays and there are limits to the amounts of endotoxin allowed in human products. Protein Sciences at Blue Sky has optimized a method for endotoxin removal which works very well to reduce endotoxin levels 30,000-fold, for example from >300,000 EU⁄mL to <10 EU⁄mL. This method involves phase separation with non-ionic detergent, whereby the endotoxin is captured in the detergent phase.

With the rise in biological therapeutic demand for large scale DNA and protein made from bacterial hosts, Blue Sky has responded with this service improvement to process both sample types and make them amenable to clients’ downstream use in animal and cell culture studies. Blue Sky bundles endotoxin removal with its endotoxin testing services.