recloning versus mutagenesis, which strategy is better

[BabcockHall]

Good afternoon,

 

We have a been working on a phosphatase, which gives indifferent over expression levels in E. coli using a T7-based system. We also have a triple mutant variant which also does not express well. Both proteins are trickier to purify than other proteins with which I have worked, because they seem prone to precipitation. We have not yet found conditions which reliably keep them in solution at concentrations above roughly 2 mg/mL. More recently we were given a plasmid with the phosphatase as a GST fusion, and it over expresses well. We are wondering what is likely to be the best way to obtain a gene for our triple mutant as a GST fusion. We see two basic alternatives:

 

A. site-directed mutagenesis of the GST fusion we already have.

B. recloning of the triple mutant that we now have in to a GST-based plasmid.

 

We are in the process of obtaining the sequence of the triple mutant but do not have it at the moment. I don't have any prior experience cloning into systems using affinity tags (my cloning skills in general are quite limited and out of date). I have consulted Amersham's GST fusion handbook (specifically Chapter 2). However, it is not very specific about how to obtain a plasmid with the insert in the correct reading from.

 

Does anyone have an opinion on which strategy is likely to be better? Does anyone have a good resource for information on cloning into GST fusion plasmids. Thank you for your time.