Reference material for this question is : J. Microbiol. Biotechnol. (2002), 12(6

Question

Reference material for this question is :  

J. Microbiol. Biotechnol. (2002), 12(6), 916–920

Purification of Recombinant Human Alpha-2a Interferon Without Using Monoclonal Antibodies

KIM, DONG CHUNG, AND JIN JUNG

Q1.

Provide a full molecular description for the procedure of cation exchange chromatography depicted in Figure 1B. Include in your answer an outline of the chemistry of both adsorption and desorption steps.

ii. Explain why this step is conducted in sodium acetate?

iii. Is good purification achieved of hIFN?-2a achieved? Explain your answer?

Q2.

Consider the step of hydrophobic interaction chromatography reported here.

i. Why do you think this step is needed?

ii. Give a molecular description of the desorption process.

iii. Why is ammonium sulphate selected to desorb material for this procedure?

iv. What is the ammonium sulphate concentration at which -The major contaminant is eluted. -The bioactive fraction is eluted.

v. What fractions correspond to the more hydrophobic species? Justify your answer.

Q3.

The report notes gel filtration (referred to as GPC) is optional but may be needed to remove bacterial protein contaminants.

Why might these still be present following the previous steps of chromatography?

Explanation / Answer

In cation exchange chromatography SP sepharose beads are used. These beads are strong anionic in nature means that maintain good range of negative charge over a wide range of charge. Generally whenever cation exchange chromatography is performed then protein should be positively charged. Therefore the pH of anion-exchange eluted protein is maintaining 5. Generally any ion exchange chromatography is conducted at low salt condition and elution is carried out by change in pH or by increasing salt concentration. At molecular level beads are negatively charged and protein is positively charged. In this chromatography generally negatively charged buffer used such as acetate therefore they do not interact with the beads and positively charged proteins will bind to these beads. At first step beads will equilibrate by the buffer and in second step protein, IFN 2-a, that present in the same buffer will introduced that allow the positively charged (cationic) protein binding. In next step loosly bound protein will be removed by the washing with equilibration buffer. In the last step a gradient of salt (sodium chloride) prepared in the equilibration buffer is applied. Due to increasing salt concentration (increase of both type of ions) will break the protein and bead interaction. In this step protein will eluted.

ii. Generally for ion exchange those buffers are preferred that having the same charge like beads. In this case SP-sepharose and acetate having same charge. One more thing that is most important is that acetate having pKa is 4.76 that maintain good buffer capacity or change is pH is hardly affected at 3.76 to 5.75 pH range. Therefore this buffer is used for the purification.

iii. In this step specific activity of the protein has been increase from 10.6 to 52.2 that shows that purify this protein by 5 fold. It is worth considering step for purification.

Q2.

Consider the step of hydrophobic interaction chromatography reported here.

i. Why do you think this step is needed?

In this step, in fig 1C, shows that it having two peaks that separated during the decrease in concentration. One more thing observed it separated the peak that come first is not showing biological activity in graph. This step is removing the unwanted protein that comes from cation exchange chromatography. Apart from that specific activity increase from 52.2 to 301 IU/mg. That is purify the protein around 6 fold.

ii. Give a molecular description of the desorption process.

I think in this question and next question adsorption will come.

  Generally in hydrophobic chromatography is performed at very high salt concentration. In high salt concentration hydrophobic amino acids get exposed. Generally in hydrophilic environment these groups are buried in the core of protein only charged residues are present at the surface. But high salt concentration will expose them and it makes an interaction with hydrophobic residues of beads.

iii. Why is ammonium sulphate selected to desorb material for this procedure?

Ammonium sulphare having very high solubility in the water and it can be used for over the great concentration which is difficult to achieve with other salts such as NaCl.

Once the salt concentration will reduce then it again get assemble at the core and that hydrophobic interaction will break down and proteins get detach from the beads and get eluted.

iv. What is the ammonium sulphate concentration at which -The major contaminant is eluted. -The bioactive fraction is eluted.

If you see the fig 1C, then find that a linear gradient is applied over 200 ml. So the concentration is falling in a linear fashion. First peak, major contaminants come approximately at 80 ml.

At zero gradient(0.5 M ammonium sulphate) is present.

So how much volume is remaining= 200-80= 120

(0.5 *120)/ 200= ~0.3 M

v. What fractions correspond to the more hydrophobic species? Justify your answer.

IIFN 2a fraction is more hydrophobic because it eluted later. So more hydrophobic force is maintained at lower salt concentration and eluted later.

Q3.

The report notes gel filtration (referred to as GPC) is optional but may be needed to remove bacterial protein contaminants.

Why might these still be present following the previous steps of chromatography?

In bioassay slight bacterial contaminated protein may interfere. In order to use in humans protein should be free from host cell protein that’s why this step was incorporated. In gel filtration chromatography only 1% -2 % sample can be use of the total column volume and this step is final polishing step. If see the table 2 this hardly make any difference in specific activity. But this chromatography cannot remove the impurities like other chromatography.

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