Protein Purification
Protein purification is a series of processes with the aim to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. For PPS, the starting point is often recombinant protein produced in the protein expression and gateway modules.
Usually, the subsequent steps in protein purification can be described as
A. Preparation of an extract
B. Bulk capture of the protein of interest (POI)
C. Intermediate purification of the POI
D. Polishing of the POI
The purification scheme used depends on the POI and its use (requirements for purity and homogeneity) and can therefore greatly vary. The preparation of an extract comprises chemical and mechanical methods, while further purification steps are mainly done by various chromatographic steps (Figure 1) using subsequently different methods e.g. affinity purification, size exclusion chromatography. Purification of the POI often includes the proteolytic removal of a tag/fusion partner to the POI. For a comprehensive overview about protein purification see the book by Burgess & Deutscher [1]
What PPS offers:
The starting material for protein purifications by PPS is mainly produced in various expressions systems in the protein expression and gateway modules. However, PPS can do protein purification from user delivered material e.g. cells, tissues or whole organisms (in the case of user provided materials, it is the users responsibility that all needed permits are provided). Protein purification at PPS can include:
- Feasibility studies of protein purification
- Preparation of extracts
- Protein purification using various schemes tailored to the POI and its use
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Characterization of the purified protein with respect to the consensus criteria for quality control of protein reagents (purity, homogeneity, identity) (https://www.nature.com/articles/s41467-021-23167-z)
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Optional: further biophysical characterizations (after discussion) are possible. This comprises for example:
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Stability of the POI in various environments and unfolding events can be analysed by differential scanning fluorometry.
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Aggregation state can be analysed by dynamic light scattering.
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Absolute molecular weight, molecular size and sample composition can be further analysed by multidetection size exclusion chromatography.
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in the case of user provided materials, it is the users responsibility that all needed permits are provided)
Figure 1.
Typical fast protein liquid chromatography (FPLC) equipment for protein purification.
[1] Guide to protein purification, 2nd edition: Academic Press; 2009.