Bio-Rad takes the stress out of protein purification
The system has, as yet, only been used on the laboratory scale, but Bio-Rad has initiated studies to check the technology’s application in biomanufacturing processes as it could dramatically reduce the number of purification steps needed.
The Profinity eXact (exact affinity cleavage technology) system promises to give protein researchers a major productivity boost by incorporating affinity purification and tag removal in a single step.
With the ever-growing demand for information about protein structure and function from proteomics researchers in drug development and biotechnology programmes there is a need for improved purification methods especially for recombinant proteins with unknown biochemical properties.
Traditional chromatographic and electrophoretic protein purification methods can be a laborious job, with significant time and effort required to develop optimal purification methods for each individual protein.
Affinity tagging methods overcome this problem by incorporating an amino acid sequence tag into the protein that will then bind with a substrate and enable the specific protein to be purified from the rest of the cell lysate.
However, the presence of the tag itself can interfere with results from protein-protein interaction experiments, functional protein studies, crystallographic and NMR (nuclear magnetic resonance) structural studies as well as in therapeutic and diagnostic applications.
Traditionally, the removal of the tags can be almost as problematic as the initial purification steps the process was designed to skip out, with inefficient cleavage by the relevant protease leading to long incubation times being needed as well as inaccurate tag cleavage leading to a mixture of products.
In answer to this problem, Bio-Rad developed the Profinity eXact fusion-tag system, which uses a mutant Subtilisin protease immobilised to a resin bead that binds the 75 amino acid Profinity eXact tag during purification. and cleaves the tag to release the native protein.
The process starts with E. coli (Escherichia coli) system that incorporates pPAL7 vectors that code for the affinity tag with the encoding sequence for the desired protein by ligation at the multiple cloning site (MCS).
The constructs are transformed into competent E. coli cells, which are cultured and the fusion-tagged protein produced.
After the cells are lysed they can be placed on the column and the tag binds to the Subtilisin with a dissociation constant (KD) of less than 100 pM, ensuring that only tagged proteins bind to the immobilised protease while the rest of the cell lysate is washed off the column.
Once the washing step has been completed, addition of a fluoride-containing buffer solution triggers the modified Subtilisin to cleave the tag directly after the 9 amino acid cleavage recognition sequence ending in ‘FKAL’.
The tag remains tightly bound to the protease while the native protein is released in purities well in excess of 90 per cent.
“Profinity eXact enables researchers to purify tag-free proteins in one hour,” said Tanis Correa, senior product manager of Bio-Rad Lab Chromatography products.
“It’s an excellent option for scientists utilising the simplicity of affinity purification, but whose applications demand unadulterated protein for functional and structural analyses.”
Once the tag free-proteins have been eluted from the column, the tag can be stripped from the protease using a weak phosphoric acid solution, regenerating the resin ready for the next sample.
According to Correa, this approach not only saves researchers from needing to use lots of often expensive protease, but it also removes the need for additional purification steps needed to separate the protein from the cleaved tag and protease.
The firm sells kits that enable the entire workflow, from the E. coli– based expression and antibody detection for checking protein expression on blots, to the affinity spin columns, cartridges and bottled media for larger scale production.
