You are harvesting an active pharmaceutical ingredient (API) from cells grown by
ID: 81491 • Letter: Y
Question
You are harvesting an active pharmaceutical ingredient (API) from cells grown by the cell culture department. You have centrifuged and filtered your product and end up with a mixed sample of six proteins. Please develop a method for the extraction of the API.
Protein
Size
Charge
Protein A
24 kD
+
Protein B
21 kD
-
Protein C
158 kD
+
Protein D *API*
28 kD
+
Protein E
123 kD
-
Protein F
27 kD
-
Size
Charge
Protein A
24 kD
+
Protein B
21 kD
-
Protein C
158 kD
+
Protein D *API*
28 kD
+
Protein E
123 kD
-
Protein F
27 kD
-
Explanation / Answer
Answer:
Based on differences in their mass, proteins can be separated by centrifugation through a solution, usually containing sucrose (an inert sugar), of increasing density called a density gradient. When mixtures of proteins are layered on top of a sucrose gradient in a tube and subjected to centrifugation, they migrate down the tube at a rate controlled by the factors that affect the sedimentation constant. The proteins start from a thin zone at the top of the tube and separate into bands, or zones (actually disks), of proteins of different masses. This density-gradient separation technique is called rate-zonal centrifugation.
Protein C and Protein E with high mass can be separated from this technique. They will be sedimented in the bottom of the tube.
proteins are exposed to the ionic detergent SDS (sodium dodecylsulfate) before and during gel electrophoresis. SDS denatures proteins, causing multimeric proteins to dissociate into their subunits, and all polypeptide chains are forced into extended conformations with similar charge:mass ratios.
Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. These gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the cross-linking reagent. Since fro rest of the proteins the size is alomost similar but their changes are different.When a mixture of proteins is applied to a gel and an electric current applied, smaller proteins migrate faster than larger proteins through the gel. The rate of movement is influenced by the gel’s pore size and the strength of the electric field.
So API protein D could be clearly separted by SDS-polyacrylamide gel electrophoresis.
We can alos apply ion exchanged chromatography after the centrifugation, a negatively charged column can be used for retaining and fractionate postively changed proteins. Here API is a positively chaged protein so we can apply this technique.
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