Precision Protein Engineering with PureFrex
The PureFrex In Vitro Translation Transcription (IVTT) system is a defined composition kit that has a much higher batch to batch consistency than cell lysate-based systems. IVTT kits are particularly useful for proteins that are hard to express in living cells, or where expression needs fine tuning with chaperones or cofactors. They are one of the fastest ways to make proteins, as the reagents just need to be mixed with the template for protein production to start.
PureFrex is assembled with tag free proteins, enabling the use of any tag on the synthesised protein without the concern of purifying the kit components.
His Tags are a popular choice to facilitate separation of the synthesised product from the reaction mix using nickel affinity, and magnetic beads (which is very efficient and suitable for high throughput methods). ReSyn Biosciences offer a wide variety of magnetic beads to help enrich or purify biomolecules, including MagReSyn NTA pre-chelated with nickel ions, or the MagReSyn NTA kit that offers 4 alternate divalent metal ions to help identify the most suitable metal ion for optimal purification of your protein.
Where nickel ions could cause issues, in downstream applications alternatives such as Strep Tag offer alternative tags with simple purification systems, however, tags need to be selected carefully, as removal can leave unwanted amino acids
2BScientific also offer a wide range of functionalised beads, microspheres and nanospheres than could help with onward application for SPR, photoacoustic imaging or drug delivery.
In some cases, the use of tags is not practical and more control over the conjugation site can be a desirable feature compared to some of the general methods used for bioconjugation.
Since PureFrex is assembled from components, custom versions of PureFrex can be made, allowing more complex protein engineering experiments that require control of particular aspects of the environment. For instance, the kit can be provided with methionine or other amino acids not included in the regular amino acid mix. This allows the researcher to add chosen amino acid analogues containing click chemistry residues, e.g. L-Azidohomoalanine, to introduce a functional group at a known location. These can potentially be detected in downstream bioorthogonal experiments for tracing and imaging with an appropriate click chemistry label, or as a targeted site for bioconjugation.
For some applications the specificity and complexity of protein engineering to this extent is not required. 2BScientific have a range of fluorophores with either classical functional groups or click chemistry motifs to help you build the compound of choice. These can permit detection, or modify protein properties. For proteins that are highly hydrophobic, the use of dPEG (homogenous PEG lengths) can help to improve protein solubility and also act as a spacer or linker in a controlled fashion.
The properties of linkers in bioconjugation can greatly affect the function of the compound. Longer linkers can counter steric hinderance issues, branched linkers can allow for higher ratios of conjugates, and linkers can be designed with cleavable sites or protected cleavable sites to allow release of a payload under specified physiological conditions. There are numerous options for linkers, cross linkers and branched linkers with homogenous numbers of PEG as spacers off the shelf, or custom structures can be made with the Biodesign service.
