Recombinant protein folding

Simon Brocklehurst Bioc smb18 at mole.bio.cam.ac.uk
Thu May 5 13:04:58 EST 1994


j.d.brinck at bham.ac.uk (Jason Brinck) writes:

>Can anyone give me some info?

>I am at the moment purifying the recombant and wild type forms of an enzyme 
>and want to analyse the differences in protein folding, if any, as the 
>recombinant form is less active than the wild type. What type of analyses are 
>there for studying protein folding and how much/how pure does the protein have 
>to be. Circular dichroism has been suggested to me.

The problem with over-expressing proteins is that some systems seem to express
the protein too quickly for it to fold _properly_.  Maybe a limiting situation 
would be getting inclusion bodies etc.  But it doesn't have to be that drastic.
So you can get "in between" situations where the protein is expressed and soluble
and purifiable, but in some way not quite correctly folded. i.e the CD and 1-D
NMR suggest that the protein is unfolded.

Don't ignore 'old-fashioned', non-sexy ignore of this - i.e. those that don't
require fancy machinery to look at the problem.

A very nice and acutally quite subtle probe of the amount of time that a protein
spends folded properly vs folded not properly is limited proteolysis.
So you would compare the behaviours of your recombinant vs wilt-type

You could do a time-course (perhaps 5 minutes, 10 minutes, 1 hour, 3 hours) with
,say, trypsin added to your protein.  Run the samples out on a gel etc.

Obviously try to pick a protease that your wild-type isn't too susceptable to!

The way proteases probably work on _proteins_ (check out the paper from Janet Thornton's group,
in the next/current issue of Protein Science) is that 5-10 residues usually need to
change conformation to fit into the active site etc i.e. these regions are flexible.

So if your recombinant protein is more susceptable to proteolysis (the bands
will disappear on the gel!!) - then it is more flexible than the wild-type,
thus spends less time in the "native" conformation etc etc.

If it works, this technique it's much less hassle than CD, or 1-D NMR!

Simon

!-----------------------------------------------------------------------
Simon M. Brocklehurst
Cambridge Centre for Molecular Recognition
Department of Biochemistry
University of Cambridge
Cambridge
UK

E-mail: s.m.brocklehurst at bioc.cam.ac.uk



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