Troubleshooting of Problems Associated with the Expression and Growth of GST-tagged Proteins

The troubleshooting guide below addresses common problems associated with the expression and growth of GST-tagged proteins.

Problem	Possible cause	Solution
No GST-tagged protein is detected in the bacterial lysate.	The culture conditions are not optimized.	Cell strain, medium composition, incubation temperature, and induction conditions can all affect yield. Exact conditions will vary for each tagged protein expressed.
	The detection method is not sufficiently sensitive.	Check for expression by immunoblotting, which is generally more sensitive than stained gels. Some tagged proteins may be masked on an SDS-polyacrylamide gel by a bacterial protein of approximately the same molecular weight. Immunoblotting can be used to identify tagged proteins in most of these cases. Run an SDS-polyacrylamide gel of induced cells and transfer the proteins to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Detect tagged protein using anti-GST antibody. Alternatively, purify the extract using Glutathione Sepharose media prior to SDS-PAGE analysis.
	Experimental error.	Select a new, independently transformed isolate and check for expression.
Most of the tagged protein is in the post-sonicate pellet.	Cell disruption is not sufficient during mechanical lysis.	Add lysozyme (0.1 volume of a 10 mg/mL lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication.
	Tagged proteins are produced as insoluble inclusion bodies.	Slow the rate of translation by altering the growth conditions Lower the growth temperature (within the range of 20 °C to 30 °C) to improve solubility. Decrease the IPTG concentration to < 0.1 mM to alter induction level. Alter the time of induction. Induce for a shorter period of time. Induce at a higher cell density for a short period of time. For more information on how to avoid formation of inclusion bodies, solubilization, and refolding, see the Challenging Protein Purification Handbook (28-9095-31).

Materials

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