Protein Purification Report: Enzyme Purification, SCIE 601, Semester 1

This report details the purification of two proteins from a default mixture using a computer simulation. The student, based on their group number (2) and birthday month (9), performed protein purification using various techniques. The first protein (9) underwent heat denaturation, ammonium sulfate fractionation, hydrophobic interaction, gel filtration, and ion exchange chromatography. The second protein (2) utilized ammonium sulfate fractionation, hydrophobic interaction with a decreasing salt gradient, anion exchange chromatography, and gel filtration. The report includes purification tables, 2D PAGE gel images, detailed instructions, and a summary of the purification strategy, explaining the rationale behind each step. The report also covers key concepts like yield, enrichment, and specific activity, demonstrating an understanding of protein purification principles and the application of various separation methods to achieve protein purity.

Running Head: PURIFICATION OF PROTEIN
Purification of Protein
Name of student:
Name of University:
Author note:

1
Purification of Protein
Purification of protein 9
The protein 9 has an enzyme activity that is stable for several hours at a temperature of 50
degrees centigrade and a pH of 4.0 to 9.5.
The protein of interest from the immunoblot image shows that it has an isoelectric pH of 6.8
and molecular weight approximately 25kDa.
The initial default enzyme mixture was subjected to heat denaturation for 20 minutes at 50
degrees centigrade.
Then further junk undesired proteins were removed with the help of 70% Ammonium
sulphate fractionation.
The hydrophobic interaction purification method was exploited to remove the proteins based
on a salt gradient of 0 to 1.0 molar. This resulted in a loss of 4% of the protein in the initial
buffer but the enzyme activity was not hampered.
The protein mixture is subjected to gel filtration exploiting Bio-gel 60 resin. This was done to
remove all the low molecular weight proteins from the default mixture. The desired protein
was eluted at the end of the elution peak and fractions were collected accordingly.
Maintaining a pH gradient 6.0 to 7.0 with pH of the buffer at 6.5 ion exchange
chromatography was exploited using Q-sepharose resin.
The gel image of the purified protein is shown below:

2
Purification of Protein
The Screenshot of the purification steps involved are:
Purification of protein 2
The protein 2 has an enzyme activity that is stable for several hours at 60 degrees centigrade
and between pH ranges of 5.0 and 10.5.
The isoelectric point of the protein is around pH 7 and molecular weight is 50Kda.
70% ammonium sulphate fractionation step was used to purify the desired protein from the bulk
protein.
Based on the hydrophobic interaction a decreasing salt gradient of 1.0-0 molar was exploited. A bulk
of junk protein was also eliminated at this step.
Anion exchange chromatography was exploited with a pH gradient of 6.5 to 7.5.
The following step involved gel filtration to purify the desired protein.
The screenshot the steps exploited is shown below:

3
Purification of Protein
The 2D gel image is given below: