Expressing GST-Fusion protein - Are larger protein inserts harder to express? (Sep/19/2005 )

Hi there,

I am trying to expression my GST-fusion protein and its mutants. I noticed that the shorter protein size mutants and GST alone expressed better (ie on comassie the bands are bigger and stained more intensed). Could it be that the bigger the protein insert, the harder it is for the BL21 DE3 to transcribe/ translate the GST-fusion protein? If so, what can I do to overcome the inconsistency? Should I induce the bigger fusion protein for longer?

Thanks for taking the time to read this. Hope to hear something soon.

Weini

It's most likely not a problem with translation, but with stability. In my lab, most fusion constructs containing GST will actually show two distinct overexpression bands on the gel - GST+fusion and GST alone. It's my guess that the extra bit gets degraded by the cell, though it's also possible that it falls off the ribosome early.

We've had some luck minimizing the presence of the GST-alone tag by reducing the temperature of induction and increasing the growth time (16 C overnight). This might help in your case.

I agree with aludlam.

Lower temperatures will improve solubility and expression on some constructs.

You might also try heat-shocking the cells for 30 mins at 42ºC before starting the induction, followed by growth at 18-24ºC

Hi there guys,

Thanks for your advices. I tried your methods but still cant get a decent expression. My suspicion is that my GST-fusioin protein is in the pellet, ie insoluble. The size of the protein is ~100kDa. If that is the case, do you have any protocol to retrieve insoluble protein from the pellet?

Thanks so much for all your help!!

Winnie

To check for insolubility you can do a simple experiment. Grow your cells (5mL is enough), pellet and do a glass bead lysis. Spin the lysate and remove the soluble fraction. Boil your pellet in denaturing buffer. Run a little of the soluble and insoluble in a gel and stain... You'll probably see your protein either in the soluble or insoluble fraction.

Use material from untransformed strain as control.

Hope it helps