Purification of soluble elastin from ovine lung: A strategy based on fast protein liquid chromatography (FPLC) : A dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Food Innovation at Lincoln University

Abstract

Sheep (Ovine) lung is a low-value by-product that contains elastin-rich connective tissue. The aim of this study was to evaluate whether sheep lung can be treated with a NaCl-NaOH-oxalic acid treatment process and purified using fast protein liquid chromatography (FPLC) to obtain soluble components rich in elastin, and to characterise the yield and composition. Fresh frozen lung tissue from healthy sheep in New Zealand was minced, homogenised in an ice bath, and washed with 1 M NaCl solution to remove soluble proteins. The remaining tissue was treated with 0.1 M NaOH solution at 95 ℃ to generate alkali resistant residue rich in elastin. Then, the residue was repeatedly extracted with 0.25 M oxalic acid solution at 95 ℃, and the extract was passed through a 10 kDa ultrafiltration step to remove salts, oxalate and other low-molecular-weight solutes before analysis and FPLC.

The initial experimental run showed a bicinchoninic acid (BCA) signal gradually increased within 0-2 hours. However, the oxalic acid extract showed no visible bands in SDS-PAGE electrophoresis, indicating that oxalate or other components interfered with the colour reaction. With 10 kDa ultrafiltration (removal of low-molecular-weight components) and other improvement measures introduced, continuous oxalic acid extraction was performed (Run 2, 40 g of fresh lung tissue), resulting in approximately 4.4 mg of BCA positive substance (≈ 0.11 mg/g wet lung tissue). Drying and grinding the alkali-resistant residue before extraction (Run 3, 80 g fresh lung tissue), and the apparent yield increased to about 20.0 mg (≈ 0.25 mg/g), with most BCA positive substances released in subsequent extraction steps.

Further purify the extracts from Run 2 and Run 3 by size exclusion FPLC using a HiPrep XK16/20 (packed with Sephacryl S-200 HR) column. A broad and trailing peak was observed at 214 nm, and the combined components captured 1.2 mg (Run 2) and 0.44 mg (Run 3) of BCA positive substance, indicating significant loss during chromatographic separation and subsequent concentration processes. SDS-PAGE electrophoresis with Coomassie Brilliant Blue and Silver Staining confirmed that both the extract after FPLC separation contains the main bands located around 75 kDa and other bands extending to above 150 kDa and around 15-50 kDa, which is consistent with the elastin derived fragments and co purified matrix proteins.

Overall, the results indicate that the NaCl-NaOH-oxalic acid treatment protocol, combined with size exclusion FPLC, can convert sheep lungs into soluble components rich in elastin. In addition, drying and grinding the residue can improve the apparent recovery rate before chromatographic separation. The preparations are not pure α-elastin, but are similar to elastin hydrolysates reported in the literature. Future work should include elastin specific quantification, better control of peptide size distribution, improved FPLC recovery and functional testing, so that this strategy can promote the value enhancement of sheep lungs as a source of elastin-based components in the field of biomaterials and nutritional supplements.