Introduction
From the previous lab, you have successfully made Escherichia coli to pick up the pGLO plasmid through the process of transformation and you have induced the expression of the green fluorescent protein (GFP) from pGLO by addition of arabinose in the growth media. Similar techniques are used in the biotechnology industry to produce several recombinant proteins such as the commercially available restriction enzyme used in the DNA fingerprinting lab. However, the protein of interest must be isolated and purified from several other proteins made by the bacteria. One of the techniques used is chromatography.
Once the bacteria have expressed the protein, the bacteria cells are concentrated by centrifugation. The cells are lysed so that endogenous bacterial proteins are released into the supernatant which is called bacterial lysate. This lysate is subjected to chromatography in order to purify the GFP protein from other proteins. GFP has many patches of hydrophobic amino acids, which collectively make the entire protein hydrophobic. Thus, hydrophobic interaction chromatography (HIC) will be used to separate GFP from other less hydrophobic and hydrophilic bacterial proteins. Chromatography is a powerful method for separating proteins and other molecules in complex mixtures. In chromatography, a column is filled with microscopic spherical beads and a mixture of proteins in a solution passes through the column by moving downward through the spaces between the beads. In HIC, the column is packed with a hydrophobic resin (beads). Based on hydrophobicity, hydrophobic substances naturally bind to other hydrophobic substances and vice versa. In HIC, the bacterial lysate is first passed through the column in a high salt buffer, a hydrophobic favor condition. The salt causes the three-dimensional structure of proteins to change so that the hydrophobic regions of the protein move to the exterior of the protein and the hydrophilic regions move to the interior of the protein. Thus, under high salt, the hydrophobic proteins will retain to the resin while less hydrophobic proteins pass through. As the salt concentration is decreased, the structure of proteins change so that the hydrophobic regions of the proteins move back into the interior and the hydrophilic move to the exterior until GFP is eluted in the last step with a no salt buffer.
Explain the relationship between genes and proteins
What is the function of lysozyme?
What was the purpose of rupturing or lysing the bacteria?
Why is HIC chosen to purify the GFP protein?