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How to purify my Recombinant protein
This example demonstrates one of the most common purification strategies:
IEX for capture, HIC for intermediate purification and GF for the polishing step.(CIPP Strategy)
The objective of this purification was to obtain highly purified protein for crystallisation and structural determination.
A more detailed description of this work can be found in Application Note 18-1128-91.
Target Molecule
Deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme.
Source Material
Recombinant protein over-expressed in soluble form in the cytoplasm E. colibacteria
Sample Extraction and Clarification
Cells were suspended in lysis buffer, 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMS, pH 7.5 and lysed using ultrasonication. Streptomycin sulphate (1%) and polyethyleneimine (0.1%) were added to precipitate DNA. The extract was clarified by centrifugation. EDTA, DTT, Benzamidine-HCl and PMSF were used in the lysis buffer to inhibit proteases and minimise damage to the oxygen sensitive-enzyme. Keeping the sample on ice also reduced protease activity.
Capture, using IEX
The capture step focused on the rapid removal of the most harmful contaminants from the relatively unstable target protein. This, together with the calculated isoelectric point of DAOCS (pI = 4.8), led to the selection of an anion exchange purification. A selection of anion exchange columns, including those from HiTrap IEX Test Kit, were screened to select the optimum medium (results not shown) before using a larger column for the optimisation of the capture step. Q Sepharose XL a high capacity medium, well suited for capture, was chosen. As shown in Figure 14, optimisation of the capture step allowed the use of a step elution at high flow rate to speed up the purification. This was particularly advantageous when working with this potentially unstable sample. |
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