Protein Expression Analysis - Fast yet Flexible

For anyone looking to automate their protein expression platform, the book “High-Throughput Protein Production and Purification” that was published in July 2019 (ISBN 978-1-4939-9624-7) is an excellent read. 

Here, various authors have contributed with their protocols for the above. A great example is Edward Kraft and his co-workers at Genentech who contributed with a chapter that describes their semi-automated method for the expression analysis of proteins from their high-throughput protein production pipeline. This platform enables the processing of multiple constructs, in small scale and in parallel in order to evaluate what construct and expression system works best for each protein in question. The result from this screening will be used in the next step, when production of the protein is scaled up.

The platform described in this chapter processes both intracellular and membrane protein, as well as extracellular (secreted) proteins. Proteins are expressed in either E.coli cells, insect cells (with BEVS) or mammalian cells. 

For intracellular proteins and membrane proteins, cells are harvested and lysed separately. For integral membrane proteins, cell membranes are solubilized. The samples are filtered and loaded onto a Beckman Biomek FX^Ρ liquid handling system PhyTip® columns with 5 μl Ni-NTA or 20 μl anti-FLAG affinity resins are used for protein purification on this platform. The approximate time for the protein purification on a 96 well plate is one hour.

For extracellular proteins, cell cultures are centrifuged and loaded onto a Hamilton STAR liquid handling system, in 50 ml conical tubes. A multiple channel pipetting head is used to aliquot the supernatant of the samples into a 96 well format. PhyTip with 10 μl Ni-NTA, 20 μl anti-FLAG or 10 μL ProPlus (MabSelect Sure) affinity resins are used for protein purification. Purification of proteins for 96 wells is about three and a half hours, including the aliquotation of the samples.

Both the Beckman Biomek FX^Ρ and the Hamilton STAR are equipped with a 96 channel pipetting head that is used for purification of the samples with PhyTip columns in the 96 well formats. Wash, equilibration, neutralization, and elution buffers are loaded onto the systems using 96 well plates and aspirated and dispensed directly by the 96 channel head and PhyTip column. The use of a 96 channel pipetting head enables high throughput purification of protein samples.

Every protein sample is purified with a PhyTip column, packed with the resin designed for the affinity tag of the protein in question. By the use of this technology, samples of the same 96-well plate can be purified with different affinity resins at the same time. Columns with different affinity resins are arranged in the tip rack so that each PhyTip column corresponds to the tag of the protein on the corresponding position in the 96 well plate. The 96 channel picks up the arranged column from the tip rack and performs the protein purification of all 96 samples simultaneously.

After protein purification, absorbance at 280 nm is used to determine protein concentration and thereby the expression level of each sample. SDS-PAGE is used for identification of full-length as well as truncated proteins, giving molar weight of protein and purity.

By this method, the expression of multiple constructs can be performed in both parallel and small scale. This enables the analysis of the conditions used for protein production, expression, lysis, purification and formulation with the target to produce a stable soluble protein. Using a 96 channel pipetting head in conjunction with PhyTip columns enables both a high throughput, and yet highly versatile platform for purification of protein samples.

Learn more about PhyTip® columns for protein purification.

Interested in learning more? Download our whitepaper - True Parallel Protein Purification with PhyTip® Columns using the Freedom EVO® and MCA96

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