Cusabio Tag-Free Recombinant
Affinity tags can interfere with various physicochemical properties and immunogenicity of recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing the promiscuous T-cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by linker dilysine and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. Recombinant protein, expressed as inclusion bodies (IBs), was purified by IB isolation, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed an emission maximum of 265 A.U. at 339 nm for the refolded protein and showed redshift in the presence of 6 M guanidine hydrochloride.
Immunization of female FvB/J inbred mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to the generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with the ZP matrix surrounding mouse oocytes. The immunized mice showed a significant reduction in fertility compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.
Label-free protein production
Compared to tagged proteins, untagged proteins have identical sequences and similar crystal structures to native proteins. Recombinant proteins with exogenous amino acids may cause immunogenicity if used in animal cells and have a limited chance of passing FDA approval. In addition, the result of functional domain and crystal structure analysis can be affected by large tags, which can limit the folding of recombinant proteins and significantly affect protein structure and biological functions.
The label-free protein service offered by Biologics International Corp (BIC) provides label-free proteins faster and more efficiently. The main advantages of our service is the certainty that there are no exogenous amino acids or proteases in our products and the elution fraction has a high homogeneity. Once we receive the DNA sequences, our dedicated teams strive to work with each customer to successfully develop the best possible tag-free protein. If you have any questions about our label-free protein service, please contact us. We will be happy to help you.
Label-free protein purification
Our tag-free protein purification service begins with tag-free gene synthesis, followed by ion exchange, which is typically the first step in the purification process. After selecting the appropriate initial conditions (eg, pH and ionic strength), elution conditions (eg, gradient volume, concentration, and slope), and matrix, we are able to purify large quantities of the target protein. Depending on the properties of the target protein, subsequent steps will include several of the following methods until we meet your specific needs.
- Ammonium Sulfate Precipitation
The solubility of proteins changes at different salt concentrations. With the increase in salt ion concentration, the solubility of proteins first increases, and then the proteins begin to precipitate. These two effects are called “salty” and “salty”, respectively. Due to ammonium sulfate’s high solubility, ability to stabilize protein structures, relatively low density, and low cost, it is especially useful as a precipitant.
- Size exclusion chromatography
Size exclusion chromatography (SEC), also known as molecular sieve chromatography, is widely applied to separate large molecules or macromolecular complexes (such as proteins). The SEC method separates different molecules by size and weight, and has several advantages, such as its good separation of large molecules from small ones, its short and well-defined separation times, and the fact that various solutions can be applied without interference. with the filtration process.
- Hydrophobic interaction chromatography
Hydrophobic interaction chromatography (HIC) separates proteins using the properties of hydrophobicity. When proteins pass through the HIC column, hydrophobic groups (such as phenyl, octyl, or butyl) attached to the stationary column can interact and bind to these proteins. The principle of protein adsorption in HIC media is complementary to that of ion exchange.