Protein Fusion and Purification System

NANTEL at UNCVX1.BITNET NANTEL at UNCVX1.BITNET
Wed Dec 9 08:05:00 EST 1992


Recently, Michael Cheng of Kyoto University asked for info on NEBs protein
fusion kit.

I have been using the fusion and purification system for the last year and,
after many modifications, I finally managed to get what I want. The new
pMal-c2 vector contains many improvements: An XmnI blunt site (which is
much easier to use than StuI), a stretch or arginines after the MBP to
increase binding to the amylose column, the LacIq repressor etc...

NEB has also released a new lysis-protein-factor-Xa-digestion buffer
that has allowed me to increase my yield to about 12 mg/l (I'm expressing
a plant transcription factor), make sure you ask NEB to send you all the
info on this new buffer. According to NEB, you should lyse the cells by
sonication. This does not have to be the case, I have modified the protocol
to use a French press since we do not have a big sonicator in our department.
Finally, the possibility of detecting recombinants by blue/white selection
and the availability of a good MBP antibody makes cloning in pMAL much
easier than in the other systems.

Unfortunately, this system is not perfect. The main problem is that your
fusion protein will be accompanied by numerous "degradation or premature-
translation-arrest" products. This makes a second purification step (I use
ion exchange) almost mandatory. Furthermore, don't believe that the MBP is
a perfectely harmless protein. We have found that the DNA-binding activity
of the fusion protein is reduced by a hundred-fold when compared to the
activity of the transcription factor after factor Xa digestion. We have
also found out that the MBPs can dimerize in the absence of their ligand
(i.e. maltose).

Hope that you are not too depressed by now, this is still a great system
as long as you know where the bugs are. If you have more specific questions,
don't hesitate to e-mail me. Cheers!

Andre Nantel
Biology
University of North Carolina-Chapel Hill
Now ranked #5 in NCAA basketball and going up



More information about the Methods mailing list