Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli
Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli
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.
Collectin, Recombinant trimeric fragment, Respiratory distress syndrome, Solubility tag, Surfactant, Surfactant protein A, Surfactant protein D, Therapeutics
151953
Watson, Alastair
9eb79329-8d32-4ed4-b8b9-d720883e8042
Sørensen, Grith L.
f0e45fe4-d4e0-4b5b-9514-45a41ab4cc51
Holmskov, Uffe
8a55aecd-694c-4d61-849b-9cf32da8ba39
Whitwell, Harry J.
94b6f20c-7924-4b33-8973-010616da9bcf
Madsen, Jens
b5d8ae35-00ac-4d19-930e-d8ddec497359
Clark, Howard
70550b6d-3bd7-47c6-8c02-4f43f37d5213
July 2020
Watson, Alastair
9eb79329-8d32-4ed4-b8b9-d720883e8042
Sørensen, Grith L.
f0e45fe4-d4e0-4b5b-9514-45a41ab4cc51
Holmskov, Uffe
8a55aecd-694c-4d61-849b-9cf32da8ba39
Whitwell, Harry J.
94b6f20c-7924-4b33-8973-010616da9bcf
Madsen, Jens
b5d8ae35-00ac-4d19-930e-d8ddec497359
Clark, Howard
70550b6d-3bd7-47c6-8c02-4f43f37d5213
Watson, Alastair, Sørensen, Grith L., Holmskov, Uffe, Whitwell, Harry J., Madsen, Jens and Clark, Howard
(2020)
Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli.
Immunobiology, 225 (4), , [151953].
(doi:10.1016/j.imbio.2020.151953).
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.
This record has no associated files available for download.
More information
Accepted/In Press date: 28 April 2020
e-pub ahead of print date: 27 May 2020
Published date: July 2020
Additional Information:
Funding Information:
This work was supported by the Medical Research Council (MRC), UK .
Funding Information:
We would like to thank Nina Kronqvist and Janne Johansson from the Karolinska Institutet for use of the NT wt and NT* solubility tags (developed and owned by Spiber Technologies) and for their technical help and advice regarding their use. We thank Nina Kronqvist for carrying out SDS-PAGE. This work was funded by the Medical Research Council (UK).
Publisher Copyright:
© 2020 The Authors
Keywords:
Collectin, Recombinant trimeric fragment, Respiratory distress syndrome, Solubility tag, Surfactant, Surfactant protein A, Surfactant protein D, Therapeutics
Identifiers
Local EPrints ID: 441033
URI: http://eprints.soton.ac.uk/id/eprint/441033
ISSN: 0171-2985
PURE UUID: aa0ccf0d-c498-4e09-bdf5-9ec7a9efc70e
Catalogue record
Date deposited: 28 May 2020 16:30
Last modified: 17 Mar 2024 03:12
Export record
Altmetrics
Contributors
Author:
Alastair Watson
Author:
Grith L. Sørensen
Author:
Uffe Holmskov
Author:
Harry J. Whitwell
Author:
Jens Madsen
Author:
Howard Clark
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics