GST Fusion Protein Purification FAQs

--- Q1: The target protein binding efficiency is low or not combined

Possible cause: error in the sequence of the protein, etc.

Solution: Sequencing confirmed, adjust the reading frame to obtain GST Tag fusion expression protein; select appropriate host bacteria, such as protease-deficient Escherichia coli, rare codon host strain Rosetta series.

Possible cause: GST fusion protein is denatured by mechanical lysis (eg ultrasound)

Solution: Excessive ultrasound heat production will destroy the tagged protein, and it is recommended to use ultra-high pressure crushing.

Possible cause: Aggregation of GST fusion protein 

Solution: Add DTT to the lysis buffer and other buffer systems. Experience has shown that 1-20 mM DTT significantly increases the binding of certain GST fusion proteins.

Possible cause: GST fusion protein is over diluted

Solution: Re-concentrate the sample to increase protein concentration.

Possible cause: The fusion protein may alter the conformation of GST, thus reducing the binding capacity of the GST-tagged protein.

Solution: Detect the binding of GST in the pGEX vector used. The cell sonic lysate with the pGEX used was prepared and tested for binding to the column. If the binding is good, it may be that the tagged protein changes the conformation of GST, thus reducing the affinity of the GST tagged protein. The results can be improved by lowering the temperature of the combination to 4 ° C to limit washing.

Possible cause: The combination of buffer conditions is not suitable 

Solution: When the pH is lower than pH 6.5 or higher than pH 8, the binding of the fusion protein to the column is insufficient, resulting in low binding efficiency. Please confirm that the magnetic beads are fully balanced by pH 6.5~8.0 buffer before being used for purification of the target protein. .

Possible cause: too many uses of the column, impurity interference

Solution: Regenerate the column or use a new column.

--- Question 2: The target protein does not elute or the elution rate is low

Possible cause: The volume of the elution buffer is too small

Solution: Increase the volume of the elution buffer.

Possible cause: insufficient time for elution

Solution: Increase the reaction time of the elution buffer with the magnetic beads.

Possible cause: The concentration of glutathione in the elution buffer is too low

Solution: increase the concentration of glutathione in the elution buffer

Possible cause: The pH of the elution buffer is too low

Solution: Adjust the pH of the elution buffer to 8-9.

Possible cause: The ionic strength of the elution buffer is too low 

Solution: Increase the ionic strength in the elution buffer. Adding 0.1~0.2M sodium chloride to the elution buffer will also improve the elution effect.

Possible cause: glutathione failure in the elution buffer is oxidized

Solution: The elution buffer is now available.

--- Question 3: There are impurities in the eluted protein

Possible cause: GST fusion protein is partially degraded by protease 

Solution: Add the protease inhibitor PMSF. Note: Serine protease inhibitors must be removed prior to the use of thrombin or factor Xa.

Possible cause: Protein is degraded in the host bacteria

Solution: Use protease-defective strain lon- or ompT, or ompT and lon double-defective strains.

Possible cause: Ultrasonic treatment when the cells are broken

Solution: Reduce the lysis time. Add lysozyme (0.1 volume of 10 mg/ml lysozyme solution, lysozyme in 25 mM Tris-HCl, pH 8.0) before mechanical lysis may make the result better. Avoid foaming as this may denature the tagged protein. Excessive cleavage also results in co-purification of host cell proteins and GST-tagged proteins.

Possible cause: The molecular chaperone may have been co-purified

Solution: Excess strips may be caused by co-purification of some molecular chaperones. These chaperones are involved in the correct folding of newly generated proteins in E. coli. Such as: DnaK (molecular weight 70, 000), DnaJ (molecular weight 37, 000), GrpE (molecular weight 40, 000) GroEL (molecular weight 57, 000), GroES (molecular weight 10, 000). Some methods for isolating GST fusion proteins from these co-purified proteins have been published.

--- Question 4: After the target protease, the electrophoresis detection found multiple bands

Possible cause: Protease cleavage occurs in the host bacteria

Solution: When the band is detected: If it is determined that the excess band is not present before the Procission Protease, thrombin, and factor Xa cleavage, these bands may be the result of degradation in the host bacteria. The target protein may contain the cutting site of Precision Protease, thrombin, and factor Xa. Please check the sequence.