Troubleshooting Cleavage Methods

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

The troubleshooting guide below addresses problems common to the majority of cleavage methods as well as problems specific to a particular method. In the latter case, the relevant method is indicated.
 

Problem Possible cause Solution
GST-tagged proteins are not cleaved completely. The ratios of PreScission Protease, thrombin, or Factor Xa to GST-tagged protein are not optimal. Check that the amount of tagged protein does not exceed the capacity of the Glutathione Sepharose chromatography medium. In most purifications, however, the medium is not saturated with tagged protein. Verify that the correct ratios of enzyme to protein are used and adjust as necessary.
For PreScission Protease and thrombin, use at least 10 units/mg of tagged protein.
For Factor Xa, use an amount equivalent to at least 1% (w/w) of the weight of tagged protein. For some tagged proteins, up to 5% Factor Xa can be used. The optimal amount must be determined empirically. In some cases, optimal results have been achieved with a tagged protein concentration of 1 mg/ml. The addition of ~0.5% SDS (w/v) to the reaction buffer can significantly improve Factor Xa cleavage with some tagged proteins. Various concentrations of SDS should be tested to determine the optimal concentration. Alternatively, increase incubation time.
The incubation time and/or enzyme concentration is not sufficient for complete cleavage of the protein from the GST tag. Increase the incubation time for the cleavage reaction. Increasing the reaction time to 20 h or more should improve cleavage as long as the tagged protein is not degraded by the extended incubation period. Alternatively, try increasing the amount of enzyme used for cleavage.
Specific cleavage sites for the proteases have been altered during cloning of the tagged protein. Verify the presence of specific enzyme cleavage sites. Check the DNA sequence of the construct and compare it with a known sequence to verify that the cleavage sites have not been altered.
The presence of cleavage enzyme inhibitors is interfering with the cleavage reaction. Remove any enzyme inhibitors that may interfere with the cleavage reaction. Prior to cleavage with PreScission Protease, buffer exchange or dialyze the tagged protein against 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5. Prior to cleavage with Factor Xa, buffer exchange the tagged protein on a desalting column (see Chapter 11, Desalting/buffer exchange and concentration), or dialyze against 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5.
Factor Xa is not properly activated. Factor Xa protease is preactivated. If using a protease from another source, activate factor Xa protease with Russell´s viper venom to generate functional enzyme. For activation of Factor Xa protease, incubate Russell´s viper venom with Factor Xa protease at a ratio of 1% in 8 mM Tris-HCl, 70 mM NaCl, 8 mM CaCl2, pH 8.0. Incubate at 37°C for 5 min.
The first amino acid after the Factor Xa protease recognition sequence is Arg or Pro. Check the sequence of the tagged protein to verify that the first three nucleotides after the Factor Xa protease recognition sequence do not code for Arg or Pro.
Multiple bands are observed after electrophoresis/ Western blotting analysis of the cleaved target protein Proteolysis is occurring in the host bacteria prior to the cleavage reaction. Determine when the extra bands appear. Verify that additional bands are not present prior to PreScission Protease, thrombin, or protease Factor Xa protease cleavage.
The tagged protein itself contains recognition sequences for PreScission Protease, thrombin protease, or Factor Xa protease. Check the sequence of the tagged protein to determine if it contains recognition sequences for the cleavage enzymes.
The tagged partner is contaminated with protease after purification Glutathione Sepharose may have been saturated with GST-tagged protein during purification. Pass the sample over a new GSTrap column or fresh Glutathione Sepharose to remove residual PreScission protease, or over a HiTrap Benzamidine (high sub) column in the case of thrombin protease or Factor Xa protease.

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