Troubleshooting in pGEX Expression Vectors

Extracted from GST Gene Fusion System, GE Healthcare, 2014

Problem Probable cause Solution
A high basal level of expression is observed Lack of catabolic repression of the lac promoter. Add 2% glucose to the growth medium. This will decrease the basal-level expression associated with the upstream lac promoter but will not affect basal-level expression from the tac promoter. The presence of glucose should not significantly affect overall expression following induction with IPTG.
No GST-tagged protein is detected in the bacterial lysate DNA sequence is not cloned in the proper translation frame in  the vector. Check DNA sequence. It is essential that protein-coding DNA sequence is cloned in the proper translation frame in the vector. Cloning junctions should be sequenced to verify that insert is in-frame. For convenience, use the pGEX 5' and 3' Sequencing Primers (see Appendix 4, Sequencing of pGEX fusions for more information on the primers). The reading frame of the MCS for each pGEX vector is shown in Figure 1.1 (pGEX vectors).

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