Early on in any recombinant protein expression and purification project, there are many factors that we at Peak Proteins consider as part of the protein construct design process. It cannot be understated the importance of committing time and effort to this stage, as the [...]
Optimising Endoglycosidase H Treatment of Proteins – All Thanks to Cauliflowers!
Peak Proteins2024-07-08T11:42:25+00:00In a previous case study, we have described the benefits of using the HEK293 suspension cell system in combination with the mannosidase inhibitor, kifunensine, and endoglycosidase H (EndoH) treatment to successfully crystallise complex proteins by removing bulky, N-linked oligosaccharides. Increasingly, we are relying on [...]
Using EDTA as a Strategy to Reduce Protein Aggregation
Peak Proteins2022-04-21T10:52:56+00:00A client requested the large-scale production of a protein that had previously been optimised for purification during feasibility studies. This protein had proven tricky to purify during the feasibility stage and two attempts had been taken to establish a suitable purification strategy. High protein [...]
Method Optimisation and Protein Production for a High Throughput Screen
Peak Proteins2022-04-21T10:53:19+00:00A client requested assistance providing protein to enable a high throughput screen (HTS). This was for an emerging target that was implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures. Their yields of purified protein from E.coli were around 1mg/L, with initial [...]
Using Peptide Mapping to Reveal Glycosylated and Non-Glycosylated Protein Forms
Peak Proteins2022-05-18T08:15:21+00:00A client had requested us to express and purify a protein for assay studies. Six 6His tagged constructs were designed to increase the chances of obtaining a suitable form of protein that could be tested in assays. After E.coli expression and nickel sepharose affinity [...]
Expression, Purification and Biotinylation of a Heterotrimeric Protein Complex
Peak Proteins2023-03-15T16:56:54+00:00A client requested the production of biotinylated and non-biotinylated heterotrimeric complex to support assay screening and biophysics. The protein complex plays a role in tumour suppression, and thus can be used to identify novel targeted therapies. Proteins A and B were cloned into [...]
Preventing Protein Aggregation to Aid Crystallisation
Peak Proteins2022-04-21T10:54:47+00:00The final purification step prior to protein crystallisation is frequently size-exclusion chromatography in the desired buffer. This enables the purification of a single oligomeric species making successful crystallisation much more likely. A frequent challenge whilst purifying proteins however is their tendency to aggregate. [...]
Generation of a broad specificity nuclease by refolding of inclusion body expressed protein. Optimisation of downstream processes after cell lysis
Peak Proteins2023-06-19T15:47:21+00:00At Peak Proteins we regularly design DNA constructs for clients, for insertion into mammalian, insect, and bacterial cell systems for over-expression of intracellular proteins. The expressed proteins then need to be isolated and purified to be supplied for a variety of uses determined [...]
Removing N-linked Oligosaccharides to Aid Protein Crystallisation
Peak Proteins2022-04-21T10:55:26+00:00A client was looking to solve the crystal structure of a secreted protein which contained a number of disulphide bonds and potential N-linked glycosylation sites, which leads to the attachment of complex sugar molecules. There was already literature available describing the expression of [...]
Time critical purification of multiple Fab constructs for assay
Peak Proteins2024-04-22T15:43:10+00:00As a leading protein purification company, BiVictriX required us to develop a method to purify 10mg each of 24 different antibody-binding fragments (Fabs) to be tested in assays. Once analysed by BiVictriX, a smaller number of chosen constructs were to be scaled up by [...]