Subtopic: Fundamentals of Downstream processing (chromatography, filtration)
Question Statement:
Two monomeric His-tagged proteins of identical molecular weight are present in a
solution. pls of these two proteins are 5.6 and 6.8. Which one of the following
techniques can be used to separate them?
Options:
(A) Denaturing polyacrylamide gel electrophoresis
(B) Size-exclusion chromatography
(C) lon-exchange chromatography
(D) Nickel affinity chromatography
Official Answer Key:
C
Step-by-Step Solution & Explanation
### **Official Solution & Concept Analysis (GATE BT 2020 Q.18)**
**1. Governing Concept**:
- **Topic**: Downstream Processing & Protein Purification Techniques
- The two monomeric proteins have:
- Identical molecular weight $\implies$ cannot be separated by **Size-Exclusion Chromatography (SEC)** or **SDS-PAGE**.
- Both are His-tagged $\implies$ both bind immobilized nickel ions, so **Ni-NTA affinity chromatography** will not resolve them.
- Distinct isoelectric points ($\text{pI}_1 = 5.6$ and $\text{pI}_2 = 6.8$) $\implies$ at a chosen buffer pH (e.g., pH 6.2), protein 1 is negatively charged ($\text{pH} > \text{pI}$) while protein 2 is positively charged ($\text{pH} < \text{pI}$).
- Therefore, they can be cleanly separated based on surface net charge by **Ion-Exchange Chromatography (IEX)**.
**2. Option Analysis**:
- **Option (A) is INCORRECT**: Denaturing PAGE separates by molecular mass, which is identical here.
- **Option (B) is INCORRECT**: SEC separates by hydrodynamic radius/mass.
- **Option (C) [Ion-exchange chromatography] is CORRECT**: Exploits the substantial difference in pI ($\Delta\text{pI} = 1.2$) and net surface charge.
- **Option (D) is INCORRECT**: Both contain polyhistidine tags and co-elute from Ni-affinity resin.
**3. Final Answer**:
- **Correct Choice**: **C**
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