The production and characterisation of an immobilised chaperonin system.
Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.
Preston
N S
NS
Division of Biochemistry, Institute of Biomolecular Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.
Baker
D J
DJ
Bottomley
S P
SP
Gore
M G
MG
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry; D018833:Chaperonins / Q000737:chemistry*; D002950:Citrate (si)-Synthase / Q000737:chemistry; D005898:Glass; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding
D018835:Chaperonin 10; D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D002950:Citrate (si)-Synthase; D000251:Adenosine Triphosphatases; D018833:Chaperonins