The purification protocol for the isolation of recombinant protein [ FldB ] with
ID: 279760 • Letter: T
Question
The purification protocol for the isolation of recombinant protein [ FldB] with pI value = 4.10. Following its overexpression and recovery from E.coli cells incorporates a two-step chromatography procedure, engaging Mono-Q matrix followed by Superdex 75.
(a) Name the specific types of chromatography which are being utilised by these two particular media (Mono-Q, Superdex 75)
(b) Explain why it is logical to isolate this recombinant protein by starting with a Mono-Q step, and then following on subsequently with Superdex 75.
(c) Suggest buffers appropriate for the MonoQ procedure:
i. Define a suitable binding (Buffer A) that would ensure that FldB adsorbs to this matrix. Justify your choice with a molecular description of events
ii. Suggest components for the elution of FldB (Buffer B). Justify your choice with a molecular description of events.
iii.How would you best monitor the purity of recovered protein samples throughout the two-step purification? Detail the underlying principles of your proposed method.
Explanation / Answer
(a) Name the specific types of chromatography which are being utilised by these two particular media (Mono-Q, Superdex 75)
Answer = The types of chromatography are Ion Exchange chromatography and Gel Filtration Chromatography which are being utilised by Mono-Q and Superdex 75 media respectively.
MonoQ is a strong anion exchanger prepacked with MonoBeads in a Tricorn columnwhich offer high resolution seperation with greater loading capacity.
Superdex 75 is a matrix used for size exclusion of gel filtration chromatography. It has a special composite matrix of dextran and agarose.
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(b) Explain why it is logical to isolate this recombinant protein by starting with a Mono-Q step, and then following on subsequently with Superdex 75.
The Mono Q step ia nothing but the ion exchange chromatography step which seperates the protein on the basis of charge and the Superdex 75 is a gel filtration chromatography step which seperates protein on the basis of mass of the protein.
Once a desirable charged proteins are seperated, the required size of protein can be seperated easily on the basis of its molecular weight.
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