Mature AGP can be purified as follows: \"AGP was purified from pooled pig serum
ID: 277591 • Letter: M
Question
Mature AGP can be purified as follows:
"AGP was purified from pooled pig serum by salting out with 350 mg ammonium sulfate added per ml of serum adjusted to pH 5.5 with 1 volume of 0.2 M sodium acetate. The supernatant was collected by centrifugation, dialyzed and subjected to ion exchange chromatography (CMSepharose) at pH 3 at low ionic strength (0.1 M sodium acetate), yielding highly enriched pig AGP in the run-through. The more basic isoforms of pig AGP could be eluted at higher pH and higher ionic strength from the same ion exchange column. The run-through fraction was further purified by chromatography on Q Sepharose at pH 5.5 at low ionic strength, followed by a polishing step on an 8100 gel filtration column. All fractions were analyzed by SDS-PAGE and/or Western blotting using a commercially available rabbit antihuman AGP antibody"
For additional information check the reference : [ Heegaard et al., 2013 ]
Q1a. Explain how ammonium sulfate fulfills the chemical requirements of a lyotrope.
Q1b.Is AGP precipitated by these ammonium sulfate conditions (350 mg/ml serum, pH 5.5)? Why, or why not?
Q1c.What is the chemical difference between the CM-Sepharose and Q-Sepharose media used in the purification protocol?
Q2a.Explain in full why it is a good plan to select CMSepharose matrix in combination with a buffer of pH 3.0 to commence AGP purification.
Q2b. Why might AGP be found in both the run-through of the CM-Sepharose column, as well as in fractions collected during elution?
Q2c.What is the purpose of the chromatography step utilizing Q-Sepharose?
Note : For additional information check the following materials : Heegaard et al., 2013 and Heegaard et al., PLoS One, 2013 .
Detail and comprehensive explanation is required.
Explanation / Answer
Q1a.
Ammonium sulfate is a perfect lyotropic salt, which is readily soluble in water and at particular ionic strength, is able to "salt-out" proteins. At such cases, the salt favors interacting with the protein rather than the water molecules of the solvent surrounding it. Infact, the cations and anions of the salt interact with the protein and the water molecules associated with the protein, increasing the solvent surface tension, decreasing the solubility of the non-polar molecules, rather favoring hydrophobic interactions and favoring the separation or salting out process. The Hofmeister series or lyotropic series is a classification of cations and anions which have the ability to salt in or out proteins. Ammonium and sulfate ions appear in the first of their respective series , explaining their chemical requirements of a suitable lyotrope. ions positioned later towards in the series promote salting in.
Q1b.
Here the aim of using the 'salting-out' methodology is to precipitate the serum proteins from the pig serum and not the protein of interest, which is AGP. The motive behind this step is to reduce the number of unrelated serum proteins by salting out with ammonium sulfate, such that following centrifugation the supernatant has the AGP protein. This partially clarified pig serum having AGP can now be subjected to ion exchange chromatographies for further purification. Therefore AGP is not precipitated by the ammount of ammonium sulfate mentioned.
Q1c.
CM-Sepharose is a cation exhanger carrying the charged group -O-CH2COO- cross-linked to agarose matrix. The serum was passed through this column, which could bind the positively charged proteins in it. To avoid local disturbances in pH, which is caused by buffering ions participating in the process of ion exchange, an eluent should be selected with buffering ions of the same charge as the substituent groups on the column's ion exchanger. SeThen the column was subjected to a low pH (pH 3) and low ionic strength buffer (0.1M sodium acetate), such that Na+ cation exhcange occurs with the column and the positively charged proteins are eluted. In this case the major AGP fraction remains in the run-through, while the positively charged proteins are eluted later. Q-Sepharose is an anion exchanger carrying the charged group -N+(CH3)3 cross linked to agarose, which can bind proteins with predominantly negative charges from the run through. The column is maintained at pH 5.5 at low ionic strength to elute the protein of interest for further processing.
Q2a.
Low pH essentially means higher H+ion concentration. Commencing the CM Sepharose purification in low pH alllows a competition between H+ ions and the positively charged proteins for binding to the -O-CH2COO- group on the column matrix. This ensures that the non-specific interactions are avoided , and the proteins with predominantly positive charges bind to the column. Non-specific binding is common when working with undefined starting material like serum and cell lysates and decrease the chances of obtaining the protein of interest, as it is lost in the initial steps when binding column matrix and subsequent washing steps.
Q2b.
As already mentioned, that the major fraction of AGP flows out in the run through without binding to the column. This is the enriched fraction. However certain basic forms of AGP bind to the column , as they are positively charged, and require higher pH and higher ionic strength to be eluted. When the elution steps for the CM Sepharose column chromatography starts , initially the positively charged proteins that bind to the column flows out. AGP proteins that were bound to positively charged serum proteins or interactors, but not making direct contact with the column matrix, will also get eluted in this process. Therefore AGP may be detected in fractions collected during elution.
Q2c.
Due to the differences in the charged groups in the mentioned chromatography columns, Q Sepharose is a strong anion exhanger , and can specifically interact with the various forms of AGP protein. Since in the run through of the CM sepharose ion exhange step, the major fraction is AGP, it is important to purify this further to get rid of the unrelated serum proteins (which had not bound to the CM Speharose) as far as possible. Q Sepharose can bind to the AGP and allow the serum proteins to flow out in the run through. Then using appropiate elution conditions AGP can be eluted from the column.
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