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Understanding the Involvement of Leukocyte Cell-derived Chemotaxin 2 (LECT2) in Amyloidosis
Linköpings universitet, Institutionen för fysik, kemi och biologi.
2019 (Engelska)Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
Abstract [en]

Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.

Ort, förlag, år, upplaga, sidor
2019. , s. 35
Nyckelord [en]
LECT2 amyloidosis, disulfide bonds, metal-binding, protein expression, refolding, purification, fluorimetry, CD, aggregation
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
URN: urn:nbn:se:liu:diva-162631ISRN: LITH-IFM-G-EX--19/3726--SEOAI: oai:DiVA.org:liu-162631DiVA, id: diva2:1377455
Externt samarbete
Scripps Research
Ämne / kurs
Kemisk biologi
Presentation
2019-09-02, 13:00 (Engelska)
Handledare
Examinatorer
Tillgänglig från: 2020-01-02 Skapad: 2019-12-11 Senast uppdaterad: 2020-01-02Bibliografiskt granskad

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Erlandsson, Lisa-Marie
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Institutionen för fysik, kemi och biologi
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