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Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli.
Immunobiology ( IF 2.8 ) Pub Date : 2020-05-27 , DOI: 10.1016/j.imbio.2020.151953
Alastair Watson 1 , Grith L Sørensen 2 , Uffe Holmskov 3 , Harry J Whitwell 4 , Jens Madsen 1 , Howard Clark 1
Affiliation  

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.



中文翻译:

在大肠杆菌中重组可溶性表达后产生新的人 SP-A 和 SP-D 三聚体片段。

新生儿呼吸窘迫综合征的表面活性剂治疗显着提高了早产儿的存活率。然而,这导致后遗症的发生率显着增加,例如新生儿慢性炎症性肺病。目前临床使用的表面活性剂制剂缺乏天然肺防御蛋白表面活性蛋白 (SP)-A 和 D。已知这些具有抗炎和抗感染特性,对于维持健康的非炎症肺部至关重要。

在生命的最初几天用这些抗炎蛋白补充目前可用的动物源性表面活性剂治疗剂可以预防早产儿炎症性肺病的发展。然而,目前用于生产重组版本的 SP-A 和 SP-D 的系统需要复杂的溶解和重折叠方案,以限制药物开发的大规模表达。

我们使用一种新的溶解度标签,描述了使用大肠杆菌表达和纯化人类 (rfh) SP-A 和 SP-D 的重组片段,而无需重新折叠。我们分别获得了每升 rfhSP-A 和 rfhSP-D 产量 23.3 (± 5.4) 毫克和 86 毫克 (± 3.5) 的平均值 (± SD)。rfhSP-D 是三聚体,68% 与 ManNAc 亲和柱结合,最终得到 57.5 mg/L 的高纯度蛋白质,大大高于通过标准重折叠方案获得的 3.3 mg/L。这种基于实验室的新方法的进一步优化可能使 rfhSP-A 和 rfhSP-D 的生产在商业上更具可行性,从而能够开发用于治疗肺部感染和炎症的新疗法。

更新日期:2020-06-23
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