Watson, A;
Sørensen, GL;
Holmskov, U;
Whitwell, HJ;
Madsen, J;
Clark, H;
(2020)
Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli.
Immunobiology
, 225
(4)
, Article 151953. 10.1016/j.imbio.2020.151953.
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Abstract
Surfactant treatment for neonatal respiratory distress syndrome has dramatically improved survival of preterm infants. However, this has resulted in a markedly increased incidence of sequelae such as neonatal chronic inflammatory lung disease. The current surfactant preparations in clinical use lack the natural lung defence proteins surfactant proteins (SP)-A and D. These are known to have anti-inflammatory and anti-infective properties essential for maintaining healthy non-inflamed lungs. Supplementation of currently available animal derived surfactant therapeutics with these anti-inflammatory proteins in the first few days of life could prevent the development of inflammatory lung disease in premature babies. However, current systems for production of recombinant versions of SP-A and SP-D require a complex solubilisation and refolding protocol limiting expression at scale for drug development. Using a novel solubility tag, we describe the expression and purification of recombinant fragments of human (rfh) SP-A and SP-D using Escherichia coli without the need for refolding. We obtained a mean (± SD) of 23.3 (± 5.4) mg and 86 mg (± 3.5) per litre yield of rfhSP-A and rfhSP-D, respectively. rfhSP-D was trimeric and 68% bound to a ManNAc-affinity column, giving a final yield of 57.5 mg/litre of highly pure protein, substantially higher than the 3.3 mg/litre obtained through the standard refolding protocol. Further optimisation of this novel lab based method could potentially make rfhSP-A and rfhSP-D production more commercially feasible to enable development of novel therapeutics for the treatment of lung infection and inflammation.
| Type: | Article |
|---|---|
| Title: | Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.imbio.2020.151953 |
| Publisher version: | https://doi.org/10.1016/j.imbio.2020.151953 |
| Language: | English |
| Additional information: | This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | Surfactant protein A, Surfactant protein D, Recombinant trimeric fragment, Collectin, Solubility tag, Respiratory distress syndrome, Surfactant, Therapeutics |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health > Neonatology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10105481 |
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