Q.43 You have expressed the following protein that has an isoelectric point of 6.0. The best
order of protein purification methodologies to obtain a pure protein is?
(A) Gel filtration chromatography, Anion exchange chromatography at pH=4.0,
Ammonium sulphate precipitation
(B) Cation exchange chromatography at pH=9.0, Ni-affinity chromatography, Gel filtration
chromatography
(C) Anion exchange chromatography at pH=8.0, Ni-affinity chromatography, Gel filtration
chromatography
(D) Ammonium sulphate precipitation, Anion exchange chromatography at pH=4.0, Niaffinity chromatography

 Introduction

Choosing the correct sequence of protein purification techniques is critical for obtaining a highly pure and biologically active protein. In competitive exams like CSIR-NET, GATE, and DBT, questions often test conceptual clarity on pI-based ion exchange chromatography, affinity tags, and polishing steps like gel filtration.

In this article, we analyze a protein:

• Expressed with an N-terminal His₆ tag

• Having an isoelectric point (pI) of 6.0

We will determine the best order of purification methodologies, followed by a clear explanation of all answer options.

 Given Information

• Protein has an N-terminal His₆ tag

• Isoelectric point (pI) = 6.0

• Goal: Obtain a highly pure protein

Key Principles to Remember

🔹 1. Charge of Protein vs pH

• pH < pI → protein is positively charged

• pH > pI → protein is negatively charged

🔹 2. Ion Exchange Chromatography

• Anion exchanger binds negatively charged proteins

• Cation exchanger binds positively charged proteins

🔹 3. Affinity Chromatography

• His₆ tag binds specifically to Ni²⁺ (Ni-NTA resin)

• Most selective → used as a major purification step

🔹 4. Gel Filtration (Size Exclusion)

• Separates based on size

• Used as a final polishing step

 Step-by-Step Logic for Best Purification Order

🔹 Step 1: Anion Exchange at pH 8.0

• pH 8.0 > pI 6.0 → protein is negatively charged

• Protein binds efficiently to anion exchange resin

• Removes many contaminants early

🔹 Step 2: Ni-Affinity Chromatography

• His₆ tag binds specifically to Ni²⁺

• High specificity → strong enrichment of target protein

🔹 Step 3: Gel Filtration Chromatography

• Final polishing step

• Removes aggregates and minor contaminants

• Ensures monodispersity

✅ Correct Answer

Option (C): Anion exchange chromatography at pH = 8.0 → Ni-affinity chromatography → Gel filtration chromatography

 Explanation of All Options

Option (A): Gel filtration → Anion exchange at pH 4.0 → Ammonium sulphate precipitation

• Gel filtration should not be used as a first step

• At pH 4.0 (< pI), protein is positively charged → won’t bind anion exchanger

• Poor logical order
  Incorrect

Option (B): Cation exchange at pH 9.0 → Ni-affinity → Gel filtration

• At pH 9.0 (> pI), protein is negatively charged

• Will not bind cation exchanger
  Incorrect

Option (C): Anion exchange at pH 8.0 → Ni-affinity → Gel filtration ✅

• Correct charge behavior

• Uses affinity step efficiently

• Gel filtration used as final polishing
  ✔ Correct Answer

Option (D): Ammonium sulphate precipitation → Anion exchange at pH 4.0 → Ni-affinity

• At pH 4.0, protein is positively charged → anion exchange unsuitable

• Ni-affinity should be before harsh precipitation steps
  Incorrect

 Exam-Oriented Takeaways

• Always compare pH with pI first

• Affinity chromatography (His-tag) is usually a core step

• Gel filtration is almost always the last step

• Avoid using size-based methods too early

Quick Rule:

Charge-based separation → Affinity → Size-based polishing