Question

Staphylococcus aureusis a bacterium causing infections in humans. In severe cases of infection in the blood, infections can be lethal. The bacterium depends on assimilating iron from the host. For this purpose, it uses a large protein complex of which one of the components (called IsdB) triggers unfolding of the heme-binding pocket of hemoglobin.  This makes it possible for the bacterium to utilize the iron from the released heme.

Following cloning of the corresponding gene, recombinant IsdB including a bioengineered series of histidine residues (His-tag) is produced in Escherichia coli. After growing the culture, the E. colicells are isolated by centrifugation, resuspended in buffer and lysed using a cell homogenizer. Following centrifugation recombinant IsdB is found in the supernatant (S). The protein is subjected to ammonium sulfate precipitation, isolated by centrifugation and redissolved in buffer. The solution of the redissolved precipitate containing the recombinant IsdB is divided in two. One half of this (A) is purified by affinity chromatography on a matrix containing immobilized nickel (II) ions, while the other half (B) is purified by gel filtration (B1) followed by affinity chromatography (B2) based on the recognition by IsdB of hemoglobin, which is immobilized on the column matrix.

The activity of IsdB is quantified (in units = U) based on a maximum in the absorbance spectrum at 410 nm seen by its reaction with heme.

B)Is method A or B to be preferred? Secondly, the specific activity of IsdB decreased after nickel (II) chromatography. Provide a suggestion what can have caused this loss. Briefly explain your answer (1-2 sentences).

Answer 1

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