Improved overexpression of Amyloidogenic peptides by solubility enhancer from spider silk

Karolinska Institutet

Huddinge

Natur Medicin

Background

Most of the data available of in vitro aggregation studies of Amyloidogenic peptides have been conducted with synthetic preparations. Synthetic preparations of amyloidogenic peptides such as Amyloid-β (Aβ) have although several drawbacks such as batch to batch variations, intrinsic impurities in synthetic preparations and is also relative expensive, especially for isotope labelling. We have recently developed a protocol for a quick, inexpensive method to produce Aβ40 and Aβ42 with superior yield compared to previous described protocols. This system is also very suitable for isotope labelling and to introduce mutations in the native sequence. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. A consecutive monomer of the NT has just recently been developed in our lab (hereafter referred to as NT*) to facilitate high expression levels and prevent aggregation of aggregation prone protein. By fusing NT* to Aβ with a proteolytic cleavage site between, expression levels were enhanced and aggregation prevented during purification. Yields of NT*Aβ fusion protein grown in M9 minimal media is comparable to the yields from LB media.

Project aims and tasks

The project aims to establish a system for overexpression of aggregation prone peptides and proteins. The first part of the project is to clone constructs of other amyloidogenic peptides such as IAPP (amylin) HD46Q (huntingtin) and α-synuclein together with NT*. After successful validation of the new constructs, recombinant overexpression and purification have to be established and optimized. Purified proteins/peptides will be characterized by various biochemical and biophysical techniques such as SDS/native PAGE, Size Exclusion Chromatography, Thioflavin T assay, CD spectroscopy, Fluorescence spectroscopy, Electron Microscopy, Mass Spectroscopy etc. Depending on success in expression and purification, the final product might also be analyzed by NMR spectroscopy.

Candidate background

Applicants should have a background in biochemistry, chemistry, molecular biology or similar field with an interest in interdisciplinary research. Particular emphasis will be placed on the ability to work in a team, strong motivation in research and creative thinking. Excellent ability to communicate in English is required.

Reference

Kronqvist N, Sarr M, Lindqvist A, Nordling K, Otikovs M, Venturi L, Pioselli B, Purhonen P, Landreh M, Biverstål H, Toleikis Z, Sjöberg L, Robinson CV, Pelizzi N, Jörnvall H, Hebert H, Jaudzems K, Curstedt T, Rising A, Johansson J: Efficient protein production inspired by how spiders make silk. Nat Commun. 8: 15504 (2017)


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