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Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli
Alastair Watson1, Grith L Sørensen2, Uffe Holmskov3
1Department of Child Health, Division of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom.
Insights
Researchers developed a new method to produce human surfactant proteins SP-A and SP-D in E. coli, potentially preventing inflammatory lung disease in premature infants. This scalable production could lead to new therapeutics for lung infections and inflammation.
Area of Science:
- Biochemistry
- Pulmonary Medicine
- Biotechnology
Background:
- Neonatal respiratory distress syndrome (NRDS) treatment improves preterm infant survival but increases chronic lung disease.
- Current surfactant therapies lack natural lung defense proteins, surfactant proteins (SP)-A and SP-D, crucial for anti-inflammatory and anti-infective lung functions.
- Recombinant SP-A and SP-D production is hindered by complex solubilization and refolding, limiting therapeutic development.
Purpose of the Study:
- To develop an efficient, scalable method for producing recombinant human surfactant proteins (rfh) SP-A and SP-D.
- To overcome limitations of current production systems by eliminating the need for refolding.
- To assess the feasibility of using these proteins in novel therapeutics for neonatal lung inflammation and infection.
Main Methods:
- Utilized a novel solubility tag for expressing and purifying recombinant fragments of human SP-A and SP-D (rfhSP-A, rfhSP-D) in Escherichia coli.
- Characterized rfhSP-D for its oligomeric state and binding affinity to ManNAc, a key component of its function.
- Quantified protein yields using the novel method and compared them to traditional refolding protocols.
Main Results:
- Achieved high yields of rfhSP-A (23.3 mg/L) and rfhSP-D (86 mg/L) without refolding.
- Confirmed rfhSP-D was trimeric and demonstrated significant binding (68%) to a ManNAc-affinity column, yielding highly pure protein (57.5 mg/L).
- The novel method significantly outperformed the standard refolding protocol (3.3 mg/L).
Conclusions:
- A novel, refolding-free method enables scalable and commercially feasible production of rfhSP-A and rfhSP-D.
- This advancement holds promise for developing new therapeutics to combat neonatal chronic lung disease, infection, and inflammation.
- Optimizing this lab-based method could pave the way for improved treatments for premature infants.
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.
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