11.22 Fractionation of cell homogenate Recombinant insulin is produced using E.

Question

11.22 Fractionation of cell homogenate Recombinant insulin is produced using E. coli fermentation. The harvested cells are homogenised using a bead mill to release the protein inclusion bodies. The cell homogenate is then treated with CNBr to cleave the cell proteins into small fragments, leaving the proinsulin as a large, uncleaved molecule. The CNBr-treated homogenate contains several components, including 5% w/w cell debris, 8% w/w proinsulin, 5% w/w cleaved proteins, and 0.5% w/w sugar. The mixture is treated using membrane filtration to remove the smaller molecules and fragments. The membrane chosen for this application gives retention coefficients of 0.95 for the cell debris, 0.90 for proinsulin, 0.25 for cleaved protein, and 0 for sugar. The effect of fouling on solute retention is negligible. The cell debris is concentrated fivefold by this process. (a) What is the composition of the retentate? (b) What is the composition of the permeate?

Explanation / Answer

Retentate is the component remain inside the membrane of gel filtration column whereas permeate because of large sized particles will pass through and would be collected first from the column. As the retention coefficient/ retardation factor is ratio of time of a molecule in stationary phase by mobile phase. Therefore retention coefficient/ retardation factor is inversely related to retention factor. Cell debris (permeate) is more retention coefficient (0.95) will show less retention and will elute first followed by proinsulin (permeate: retention coefficient 0.90). The cleaved proteins and sugars with very low retention coefficient (0.25 and 0 respectively) will be retained in retenatate and will not be eluted or may be eluted very late.

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