Recombinant synthesis of pulmonary surfactant proteins SP-B and SP-C

Student thesis: Doctoral ThesisDoctor of Philosophy

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Authors

Anjana Patel

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Abstract

SP-B and SP-C are two pulmonary surfactant proteins. They were isolated in the tear film in 2006 (Lukovic et al. 2006).The molecular mechanism governing lipid spreading in the tear film is not well understood and, along with their production in yeast (as an alternative to animal derived material) provide the motives for this research.To investigate the involvement of SP-B and SP-C in lipid spreading, SP-B and SP-C have been produced as recombinant proteins in Pichia pastoris yeast. SP-B and SP-C are cleaved from their proproteins to produce mature active proteins and both forms were produced recombinantly. The proteins were extracted with detergents, polymers and organic solvents to determine the best method of protein isolation and purification. Purified proteins were subsequently tested for surface activity using a Langmuir trough; proSP-C10 and SP-B demonstrated surface tension activity.The mechanism of SP-B and SP-C was examined through generation of ab initio structural models. The behaviour of SP-B and SP-C was observed in membranes based on phospholipid compositions surrounding the SP-B and SP-C in the yeast plasma membrane using mass spectrometry analysis.Computational modelling then demonstrated that SP-B induces lipid curvature, whilst SP-C may help to stabilise lipid vesicles by forming a molecular bridge between the membrane and vesicle.SP-B is thought to help remodel the lung lipid layer on compression through the formation of vesicles that are connected to the membrane by SP-C.

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Original languageEnglish
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Award date20 Sep 2018

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