Protein purification is a critical step in producing recombinant proteins for research, industrial, or therapeutic applications. The goal is to isolate the target protein from a complex mixture of other proteins and impurities. Here are some common protein purification methods:

Protein Purification Methods

     1.  Affinity Chromatography:

          ● Principle: Utilizes a specific interaction between the target protein and a ligand that is immobilized on a chromatography matrix.

          ● Common Applications: His-tag, GST-tag, FLAG-tag purification.

          ● Advantages: High specificity, can achieve high purity in a single step.

 

     2.  Ion Exchange Chromatography:

          ● Principle: Separates proteins based on their net charge at a given pH. Cation exchange chromatography uses negatively charged matrices to bind positively charged proteins, while anion exchange chromatography uses positively charged matrices to bind negatively charged proteins.

          ● Common Applications: Purification of proteins with different isoelectric points.

          ● Advantages: Can handle large volumes, good for separating proteins with small differences in charge.

 

     3.  Size Exclusion Chromatography (SEC):

          ● Principle: Separates proteins based on their size and shape. Smaller proteins enter the pores of the chromatography media and elute later, while larger proteins are excluded from the pores and elute earlier.

          ● Common Applications: Desalting, buffer exchange, final polishing steps.

          ● Advantages: Gentle on proteins, maintains protein activity.

 

     4.  Hydrophobic Interaction Chromatography (HIC):

          ● Principle: Separates proteins based on their hydrophobicity. Proteins bind to hydrophobic ligands on the chromatography matrix in high salt conditions and are eluted by decreasing the salt concentration.

          ● Common Applications: Purification of proteins with hydrophobic regions.

          ● Advantages: Can be used to remove aggregates and denatured proteins.

 

     5.  Metal Chelate Affinity Chromatography (IMAC):

          ● Principle: Utilizes the affinity of histidine residues for metal ions (e.g., Ni²⁺, Co²⁺) that are immobilized on the chromatography resin.

          ● Common Applications: His-tagged protein purification.

          ● Advantages: High specificity, efficient for His-tagged proteins.

     6.  Reverse Phase Chromatography (RPC):

          ● Principle: Separates proteins based on their hydrophobicity using a non-polar stationary phase and a polar mobile phase.

          ● Common Applications: Purification of peptides and small proteins.

          ● Advantages: High resolution, useful for analytical purposes. 

Affinity chromatography with specific tags like His-tag, GST-tag, or FLAG-tag is commonly used for its high specificity and ease of use. Other methods such as ion exchange, size exclusion, and hydrophobic interaction chromatography are often used in combination to achieve high purity and activity of the target protein.

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial, yeast, insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.