Subtopic: Fundamentals of Downstream processing (chromatography, filtration)
Question Statement:
A recombinant protein is secreted extracellularly in soluble form by an E. coli
culture. Which of the following downstream processes is/are involved in the
purification of the extracellular secreted protein?
Options:
(A) Cell disruption
(B) Membrane ultrafiltration
(C) Solubilisation of inclusion bodies
(D) Liquid chromatography
Official Answer Key:
B;D
Step-by-Step Solution & Explanation
### **Official Solution & Concept Analysis (GATE BT 2025 Q.27)**
**1. Governing Concept**:
- **Topic**: Downstream Processing & Primary Recovery Operations
- For a **soluble extracellular recombinant protein** produced by *E. coli*:
- The target protein is already present in the liquid fermentation broth (culture supernatant).
- Primary recovery requires solid-liquid separation to remove intact bacterial cells without releasing intracellular contaminants.
- **Centrifugation** or **Microfiltration** separates the biomass from the liquid, retaining the soluble protein in the clarified supernatant/filtrate.
- **Cell Disruption** (sonication, high-pressure homogenization, French press) is **unnecessary and harmful** because it lyses host cells, releasing host cell proteins (HCPs), genomic DNA, and endotoxins into the product stream.
**2. Option Analysis**:
- **Option (A) [High-pressure homogenization of cells] is INCORRECT**: Used for intracellular products; lysing cells here severely contaminates extracellular product with host cellular debris.
- **Option (B) [Centrifugation to harvest clarified supernatant] is CORRECT**: Rapidly pellets bacterial cells, yielding clean supernatant containing the soluble target protein.
- **Option (C) [Sonication to lyse cells] is INCORRECT**: Unwanted lysis that degrades and contaminates extracellular product.
- **Option (D) [Microfiltration using a 0.2 µm membrane] is CORRECT**: Standard tangential flow microfiltration retains *E. coli* cells ($~1–2\text{ }\mu\text{m}$) while allowing soluble protein to pass freely into the permeate.
**3. Final Answer**:
- **Correct Options**: **B, D**
Practice This in Real CBT Exam Interface
Test yourself with the official TCS-iON palette, virtual calculator, and immediate performance analytics.