While we like the expression, refolding and formulation of the relatively small and simple proteins like most chemokines and cytokines, we also get excited at the site of more challenging projects that our clients bring to us! Examples of these include the expression and lyophilisation of large proteins such as Cas9 with 161-kDa mass, or the refolding and stabilisation of recalcitrant proteins exemplified by the IGF-binding proteins with nine disulfides bonds, or even forcing E. coli to express proteins that are just difficult like some antimicrobial chemokines.
Representative examples of the proteins that we expressed to date are shown next. If the proteins that you are interested in are similar to what you see in this list, then we are in the prime position to express and/or analyse these proteins for you. Please contact us and we may even have a sample of these proteins for you to assess in your own lab. We will be also more than happy to discuss any other expression projects for proteins that are not in this list.
However, please note that our services and products are only for scientific research and certain industrial application, and not available to the public.
The chimeric Cas9-NLS includes a wild type S. pyogenes Cas9 followed by a C-terminal nuclear localisation signal (NLS: SV40) and a His tag. The protein was lyophilised, giving it the following advantages:
The Cas9-NLS was verified to have the expected structure and biological activity. In a representative experiment, 800 µg of a 1,100-bp PCR product was cleaved with 0.5 µg of the Proteowa's NLS-Cas9 as well as those from certain commercial manufacturers. The digested products were then resolved on Agarose gels and visualised. The sub-optimal amount of Cas9 was intentional to avoid complete digestion of the substrate, hence proper comparison of the enzyme efficiencies. Results shown in the gel image below confirms that, if not better, lyophilised Cas9-NLS is as active as the other enzymes.
1- Control (DNA only)
2- Treated by Proteowa Cas9
3- Treated by vendor A Cas9
4- Treated by vendor B Cas9
5- Treated by vendor C Cas9
Note that because the nuclease activities of Cas9-gRNA complexes vary greatly, it is essential that the activity of any ribonucleoprotein is confirmed in vitro before any in vivo transformation experiment. For the in vitro assay of gRNA, we recommend applying a molar ratio of 10:12:1 (Cas9:gRNA:target DNA). The following calculator may be used to work out the amounts of Cas9 and gRNA that is needed for your DNA sample.
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All our chemokines are expressed as fusion proteins that are then cleaved from the fusion partner to ensure they have the exact N-terminal sequence as the wide-type protein. We have shown that our chemokines have the exact expected sequences, formed the right disulphide bonds and has the correct structures and biological activities. The proteins are purified to at least 95% and sterilized before further steps. When required, they are also lyophilised under sterile conditions.
Our cytokines are expressed and analysed by the same way as the above chemokines.
This is one of our ongoing projects. IGFBPs are readily expressed in E. coli but the resulting inclusion bodies are hard to refold. We hypothesize that this is the case because the proteins (1.) lack the glycosilations normally found in the wide-type proteins, (2.) contain multiple cysteines, and (3.) have a natural tendency to interact with other proteins (suggesting the presence of exposed hydrophobic patches). Nevertheless, we have recently folded these proteins and showed that they form monomers with a expected melting curved and a melting temperature of 42.5°C under the experimental conditions.
All our ubiquitin-specific proteases were readily expressed in folded/soluble forms in E. coli.