Designing a high throughput refolding array using a combination of the GroEL chaperonin and osmolytes.
Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL-GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin-substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a 'folding array' to identify superior folding conditions. The stable chaperonin-substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (approximately mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.
Voziyan
Paul A
PA
Department of Medicine/Nephrology, Vanderbilt University Medical Center, Nashville, TN 37232-2372, USA.
Johnston
Mary
M
Chao
Angela
A
Bomhoff
Greg
G
Fisher
Mark T
MT
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D008863:Microspheres; D017510:Protein Folding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013395:Sucrose / Q000378:metabolism
D018834:Chaperonin 60; D011506:Proteins; D013395:Sucrose; D000255:Adenosine Triphosphate; D001120:Arginine; D005990:Glycerol