Expression And Purification Of Recombinant Linker Histone H1a21c Proteins From Escherichia Coli

722 words - 3 pages

Two restriction enzymes, Nde1 and Xho1, enabled the gene coding for the H1a21c protein to be successfully inserted into the pET22b plasmid. However, since the pET22b vector contains a His- tag sequence, it was important that the Xho1 enzyme to introduce a stop codon that prevented a His- tag to be attached to the H1 protein.
The SP FF ‘fast S’ column was used to purify the H1 protein based on the ionisation state of the protein. Referring to the structure of H1 proteins, they contain a high level of lysine residues (compared to core histones), thus making them relatively basic (Johns, 1971; Cole, 1984). The cysteine residue increased this positive nature of the H1 protein, allowing the ...view middle of the document...

This was a crucial step in order to purify the H1a21c protein further. Hydrphobic interaction chromatography promotes protein separation by interacting hydrophobic ligands and non- polar regions on the protein (Melander and Horva´th, 1977; Fausnaugh and Regnier, 1986; Roe, 1989). Hydrophobic interactions occurred between the H1 protein and the phenyl ligand on the phenyl sepharose column.
An initial high salt concentrated buffer enhanced the adsorption of the H1 protein by further exposing its hydrophobic residues (Melander and Horva´th, 1977; Fausnaugh and Regnier, 1986; Roe, 1989). In order to elute the H1 protein, a decreasing concentrated salt buffer was used in order to de- expose the hydrophobic residues, and this was confirmed by the decrease of conductance (Figure 5) as the proteins eluted.
A single defined peak (Figure 5) represented the elution of the H1 protein together with few contaminants. TCA precipitation was conducted in order to precipitate and concentrate the H1 protein for further SDS- PAGE analysis. It is important to note that the TCA precipitation method does not favour the precipitation of unfolded proteins (Rajalingam et al., 2009). The SDS- PAGE gel showed a more purified H1 protein, as seen by three distinct bands...

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