| Title: |
Evaluation of refolding conditions for a human recombinant fusion cytokine protein, promegapoietin‐1a |
| Authors: |
Boyle, Denis M.; Johnson, Gary V.; Heeren, Robert A.; Shell, Robert E.; Banerjee, Amit; Gustafson, Mark E. |
| Source: |
Biotechnology and Applied Biochemistry ; volume 49, issue 1, page 73-83 ; ISSN 0885-4513 1470-8744 |
| Publisher Information: |
Wiley |
| Publication Year: |
2008 |
| Collection: |
Wiley Online Library (Open Access Articles via Crossref) |
| Description: |
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. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| DOI: |
10.1042/ba20070069 |
| DOI: |
10.1042/BA20070069 |
| Availability: |
http://dx.doi.org/10.1042/ba20070069; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1042%2FBA20070069; https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1042/BA20070069 |
| Rights: |
http://onlinelibrary.wiley.com/termsAndConditions#vor |
| Accession Number: |
edsbas.6BA96296 |
| Database: |
BASE |