Evaluation of refolding conditions for a recombinant human interleukin-3 variant (daniplestim).
The refolding of daniplestim, a human interleukin-3 variant (SC-55494) from Escherichia coli inclusion bodies, was optimized using a reversed-phase HPLC method developed to permit quantification of the reduced and oxidized forms of daniplestim. The presence of cysteine or dithiothreitol accelerated refolding of daniplestim from E. coli inclusion body slurries dissolved in urea or guanidine solutions and was complete in 4-6 h. Regardless of the dissolution and refolding protocol used to renature daniplestim, equivalently bioactive protein was produced. Under refolding conditions, no covalent modification of daniplestim by cysteine or cyanate was observed. The folding process was characterized further by following the unfolding of purified daniplestim by far-UV CD and fluorescence spectroscopies under both oxidizing and reducing conditions at pH values between 7 and 11. Formation of the single disulphide bond had a large stabilizing effect on daniplestim structure ( approximately 4-5 kCal at pH 9.5). This thermodynamic stabilization drove the refolding process towards the native form, even under conditions where the reduced protein was largely unfolded. From these data, scaleable refolding conditions for daniplestim were established.
Boyle
D M
DM
Department of BioProcess Technology, Searle R&D/Monsanto Co., 700 Chesterfield Village Pkwy., St. Louis, MO 63198, USA.
McKinnie
R E
RE
Joy
W D
WD
Violand
B N
BN
McLaughlin
J K
JK
Landis
B H
BH
D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003956:Dialysis; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007377:Interleukin-3 / Q000819:agonists* / Q000737:chemistry* / Q000378:metabolism / Q000031:analogs & derivatives; D010446:Peptide Fragments; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D004220:Disulfides; D007377:Interleukin-3; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C093340:daniplestim; D014508:Urea; D003545:Cysteine