A study of coacervation of recombinant proteins in yeast with microscopic technologies
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Journal Title
Journal ISSN
Volume Title
Kemian tekniikan korkeakoulu |
Master's thesis
Authors
Date
2019-12-17
Department
Major/Subject
Biotechnology
Mcode
CHEM3022
Degree programme
Master's Programme in Chemical, Biochemical and Materials Engineering
Language
en
Pages
72
Series
Abstract
Liquid-liquid phase separation, also known as coacervation, of biomolecules has long been considered as a key role in the formation of membrane-less compartments. Moreover, coacervation of proteins has become increasingly recognized and understood in nature as an intermediate step toward the assembly of biopolymeric materials, such as mussel adhesive and elastin. Due to the excellent properties and wide applications of biomaterials, there is currently a great impetus in producing protein-based materials using recombinant DNA technologies. Coacervation of recombinant proteins into high-concentrated droplets is a hallmark feature of materials with biological functions. However, identification of self-coacervating proteins requires purification of all tested proteins and reconstitution of coacervates in vitro, which is ineffective to find novel proteins that show condensation. An efficient alternative to identify recombinant proteins that can lead to material formation is brought up in this thesis, which is to characterize the physical properties shown by coacervates formed in living cells by using microscopic technologies. Only protein candidates that form in vivo coacervates with favorable properties would be selected for further in vitro characterizations. Therefore, the goal of this thesis is to explore the feasibility of this alternative to select self-coacervating proteins for materials based on the physical properties showed by in vivo coacervates. In this thesis, a model protein, Saccharomyces cerevisiae Sup35 which can form reversible condensates upon transient stresses, was used as a model protein. Two disordered elements, the NM domain from Sup35 and the ADF3 sequence from spidroin, were used to construct multiple fluorescent fusion proteins that were predicted to be able to undergo coacervation. Those recombinant proteins were produced in Saccharomyces cerevisiae with different levels, and the properties of their resultant coacervates were mainly characterized by fluorescence microscopy, super resolution radial fluctuations, time-lapse imaging. The experiment results showed the properties of in vivo coacervates are related to the features of proteins and the difference of properties exhibited by different coacervates can be distinguished by microscopic technologies, thus suggesting that primary selection of self-coacervating proteins in vivo provides us a new strategy to develop new protein-based materials.Description
Supervisor
Linder, MarkusThesis advisor
Osmekhina, EkaterinaKeywords
liquid-liquid phase separation, intrinsically disordered domain, biomaterial, silk-like protein, microscopic technologies, coacervation