Reconstitution of Connexin-43 in Artificial Membranes

by Yeimar Portillo
Doctoral thesis
Date of Examination:2022-03-02
Date of issue:2022-04-01
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Michael Meinecke
Persistent Address: http://dx.doi.org/10.53846/goediss-9162

 

 

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Abstract

English

Synthetic biology aims to engineer complex biological functions in an artificial way. A variety of molecular tools and techniques allow mimicking of processes of living systems in a synthetic way. In that context, the production of compartments with a minimal and sufficient number of components to sustain life has proven relevant in recent years 1. Cell-free systems (CFS) are powerful platforms for the expression of proteins. In combination with membrane mimic structures, CFS can be used to synthetically reproduce processes like metabolism, transport of molecules, and communication between compartments. Connexin 43 is a transmembrane protein involved in direct cell-to-cell communication pathways in many cell types. In this project, a CFS was used to reconstitute Cx43 in artificial membranes as a first step in the direction of mimicking intercellular communication. Functional plasmids compatible with CFS were produced to express EGFP-Cx43, which allowed monitoring samples via fluorescence. In vitro protein expression took place in the presence of vesicles of L-α-PC: DOTAP: TxR in a cotranslational manner. The presence of Cx43 in vesicles was evaluated by Western Blot and fluorescence spectroscopy, reporting that ~ 50-80 % of the protein variants inserted into the vesicles. The incorporation of EGFP-Cx43 into vesicles was affected by the presence of EGFP as it made them less hydrophobic than the WT Cx43. Additionally, the incorporation of EGFP-Cx43 into GUVs and its analysis by CLSM was successful. The model system presented in this work could be further optimized and used to establish communication pathways between artificial minimal cells.
Keywords: cell-free expression; Connexin 43; synthetic biology; bottom-up approach
 
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