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Escobia
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Protein # 15
= Enzymatic ability is stable for several hours at temperatures up to forty degrees Celsius and
pH between 4.5 and 9.5
Initial mixture:

1st step: Ammonium Sulfate fractionation

Yield: 100 %, Enrichment: 1.8, Cost: 0.065

For this initial step done for purifying protein 15, 50 % saturation of Ammonium Sulfate was
used. The step was chosen as it is a cheap process that has no adverse effects on the enzymatic
activity of the protein to be purified thus providing the opportunity for a higher yield. The
saturation was set at 50% as it is the value that will precipitate the highest amount of protein
(referring to undesired proteins present in the solution) without precipitating any of the
enzyme (desired protein). Furthermore, this initial step is useful to quickly remove large
amounts of contaminant proteins and other molecules which will make succeeding
purification steps easier.

Gel filtration
Yield:99.3%, Enrichment : 9.9 , Cost: 0.292
Several proteins are present in the mixture given for purification and each one with
corresponding molecular weight as indicated in the 2D PAGE image. The specific gel used
was Sephacryl S200 HR. This step was done to first separate proteins based on molecular
weight before separating proteins using other properties. Gel filtration was used to obtain a
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fast, high recovery separation of the proteins. In addition, Sephacryl has high chemical
stability and tolerance of high flow rate. Sephacryl has an approximate fractionation range for
peptides and globular proteins of between 5,000 to 250,000 (in molecular weight). Sephadex
is a good media to use for the process as it has a wide range for fractionation which matches
the span of molecular weights of proteins present in the mixture.

As seen below in the graph of the enzymatic activity of different fractions derived from gel
filtration, there is a clear separation of the proteins by their molecular weight. Larger proteins
pass through the column faster than smaller proteins. The desired protein seems to be an
intermediate in terms of molecular weight and is eluded and collected in fractions 45 to 60
based on enzymatic activity assay. By pooling the fractions with high enzymatic activity only,
other undesired proteins are removed resulting to a mixture of proteins containing the desired
protein 15 and proteins that are at a closer range of molecular weight to protein 15 and few
with very high molecular weights.
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Ion exchange chromatography

Yield: 99.3% , Enrichment: 16.5 , Cost: 0.520

This test is done to exploit the net charge of the desired protein. In this test, the isoelectric
point of the desired protein is important. This was determined using 2D PAGE showing the
molecular weight of the proteins and their isoelectric point. Protein 15 has an isoelectric point
between ph 7 to 8 (in the process assumed to be approximately 7.2). The buffer was set to pH
6.2 , one pH level lower than the protein 15`s pI. The starting pH also is effective for the
chosen media which has a range of pH 5 to 9 as effective starting pH. Salt gradient was used
set at 0.2 molars to 0.8 molars. Positively charged proteins will bind to CM groups and can
subsequently be dislodged by solutions with higher salt concentrations or a higher pH. The
use of a salt gradient to increase the ionic strength of the column buffer reduces interaction
between the ion exchanger and the protein causing the later to elute.
Thus, in this process, the proteins that had a lower pI were eluded in the earlier collected
fractions. The separation is due to the increasing concentrations of salt used for the gradient.
Less positively charged proteins are eluded earlier because they are dislodged the use of less
cocnentrated salt. While, proteins which are more positively charged (in this case, the desired
protein) are eluded much later because a higher concentration is needed to dislodge the
protein.
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Summary table of steps and SDS PAGE after all purification steps
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Summary Table of Steps for the Purification of Protein 15.

2D PAGE of pooled fractions acquired after all purification steps.