Evaluation of refolding conditions for a human recombinant fusion cytokine protein, promegapoietin-1a.
Conditions to obtain correctly folded PMP-1a (promegapoietin-1a), an engineered fusion IL-3 (interleukin-3) and thrombopoietin receptor agonist from recombinant Escherichia coli IBs (inclusion bodies), were defined to generate sufficient amounts of protein for evaluation as a potential therapeutic compound. Several ionic and non-ionic detergents, as well as the chaotrope urea, in combination with selected additives, were screened for their ability to dissolve IB protein and promote formation of monomeric, oxidized protein. Upon dissolution, soluble aggregates constituted 50-60% of total protein in detergent-solubilized IBs depending on the level of detergent used, whereas use of urea increased aggregation to approx. 70%. Subsequent addition of 5 mM cysteine or DTT (dithiothreitol) reduced the levels of aggregation, but never lower than approx. 20%. Refolds from detergent-solubilized IBs with or without organic modifiers characteristically produced multiple persistent misfolded species. However, the addition of a 12:1 molar excess of cystine (cystine/DTT) to urea-dissolved IBs containing DTT, followed by dilution, promoted the formation of correctly oxidized, disulfide-paired PMP-1a monomer with minimal misfolds present. Thus treatment of urea-dissolved proteins with thiol-group-containing additives and control of dilution, pH, protein concentration and order of addition were able to produce a maximum refold efficiency of 40-50% of correctly paired protein monomer.
Boyle
Denis M
DM
Department of Bioprocess Research and Development, Pfizer Global Biologics, Pfizer Corp., 700 Chesterfield Village Parkway, Chesterfield, MO 63017, USA. denis.m.boyle@pfizer.com
Johnson
Gary V
GV
Heeren
Robert A
RA
Shell
Robert E
RE
Banerjee
Amit
A
Gustafson
Mark E
ME
D006801:Humans; D007377:Interleukin-3 / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013926:Thrombopoietin / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*
D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; C438891:promegapoietin-1a; D013926:Thrombopoietin