Thesis defense: Florent Fontaine
Lipid Droplet
recombinant protein
N. benthamiana
C. sativa
plant biotechnology
Thursday, January 30th, 1:30 pm - INRAE, Versailles
Functional study of Lipid Droplet (LD) targeting domains to improve the purification of recombinant proteins
The production of hydrophobic recombinant proteins, such as transmembrane proteins, remains a challenge due to their association with a lipid environment, making purification complex and costly (up to 80% of production costs). An innovative approach was proposed in this thesis: using the hydrophobic properties of the plant lipid droplet (GL) to enable the folding and flotation purification of membrane proteins, thanks to anchoring via oleosin, a major protein in seed GLs. Although this technique has been validated for soluble proteins such as insulin, it has not yet been applied to transmembrane proteins. GLs are dynamic structures formed by a core of neutral lipids, such as triacylglycerols (TAGs), surrounded by a monolayer of phospholipids to which proteins may be associated, mainly from the ER or cytosol. Some of these proteins, involved in GL biogenesis, attach early to their surface. Although the proteins do not possess a conserved domain for GL addressing, their localization often depends on specific structural motifs. However, these motifs may also be present on proteins not associated with GLs, complicating the study of their specificity and highlighting the need for further research in this area. This thesis contributes to elucidating the mechanisms governing interactions between proteins and GLs, focusing on factors influencing their specificity and affinity for the GL surface, with the aim of identifying potential levers for biotechnological applications. In this study, I was able to demonstrate that GLs can be exploited for the purification of hydrophobic proteins fused to AtOLE1, the major oleosin of Arabidopsis thaliana seeds, using the E and M proteins of SARS-CoV-2 as proof of concept. To do this, I first used Nicotiana benthamiana transiently overproducing GLs and microscopy to assess whether E and M were found at GLs through AtOLE1. In addition, I developed a colocalization pipeline that enabled me to quantify this specificity, i.e. their ability to target GLs exclusively, and thus show that AtOLE1 was indeed able to address E to GLs. E and M were then expressed in Camelina sativa seeds. Once the GLs had been purified, it was possible to detect E and M on the GL surface, and that this addressing was enhanced by fusion with OLE1.
In order to improve our knowledge of the mechanisms of interaction between proteins and GLs, I assessed the protein/GL interaction specificity of a collection of proteins and domains by microscopy, using the same procedure I set up for membrane proteins in N. benthamiana. In addition, this specificity was compared with the structural properties of the proteins, such as charge and hydrophobicity. Unexpectedly, no correlation was found between these structural properties and GL specificity. Instead, it seems that this specificity is more influenced by the function of the proteins in GL biogenesis or by their arrival kinetics. Indeed, proteins that localize early on the surface of the monolayer show increased specificity. On the other hand, the affinity of proteins to GLs, i.e. the ability to remain associated with GLs, could not be quantified in this chassis, as the transient overproduction of GLs was not sufficient for this. A new chassis, a N. benthamiana stably overaccumulating GLs, therefore had to be produced. Compared with the WT, this chassis has 22 to 23 times more GLs. After isolation, these GLs were subjected to various washes, with increasingly stringent conditions, and the associated proteins were detected by biochemical techniques. This affinity assessment technique, initially based on the seed, was used for the first time in a leaf context. The results obtained for three proteins showed that the proteins were present as oligomers on the GLs and remained bound regardless of the stringency of the treatment. The results obtained previously were validated in C. sativa seeds, by tracing the production of a commercial growth factor protein, HsFGF2. These observations highlight that the arrival kinetics and functional role of proteins in GL biogenesis are determining factors for their specificity and affinity in GL addressing. This knowledge will help optimize the use of GLs and their associated proteins for biotechnological applications.
The production of hydrophobic recombinant proteins, such as transmembrane proteins, remains a challenge due to their association with a lipid environment, making purification complex and costly (up to 80% of production costs). An innovative approach was proposed in this thesis: using the hydrophobic properties of the plant lipid droplet (GL) to enable the folding and flotation purification of membrane proteins, thanks to anchoring via oleosin, a major protein in seed GLs. Although this technique has been validated for soluble proteins such as insulin, it has not yet been applied to transmembrane proteins. GLs are dynamic structures formed by a core of neutral lipids, such as triacylglycerols (TAGs), surrounded by a monolayer of phospholipids to which proteins may be associated, mainly from the ER or cytosol. Some of these proteins, involved in GL biogenesis, attach early to their surface. Although the proteins do not possess a conserved domain for GL addressing, their localization often depends on specific structural motifs. However, these motifs may also be present on proteins not associated with GLs, complicating the study of their specificity and highlighting the need for further research in this area. This thesis contributes to elucidating the mechanisms governing interactions between proteins and GLs, focusing on factors influencing their specificity and affinity for the GL surface, with the aim of identifying potential levers for biotechnological applications. In this study, I was able to demonstrate that GLs can be exploited for the purification of hydrophobic proteins fused to AtOLE1, the major oleosin of Arabidopsis thaliana seeds, using the E and M proteins of SARS-CoV-2 as proof of concept. To do this, I first used Nicotiana benthamiana transiently overproducing GLs and microscopy to assess whether E and M were found at GLs through AtOLE1. In addition, I developed a colocalization pipeline that enabled me to quantify this specificity, i.e. their ability to target GLs exclusively, and thus show that AtOLE1 was indeed able to address E to GLs. E and M were then expressed in Camelina sativa seeds. Once the GLs had been purified, it was possible to detect E and M on the GL surface, and that this addressing was enhanced by fusion with OLE1.
In order to improve our knowledge of the mechanisms of interaction between proteins and GLs, I assessed the protein/GL interaction specificity of a collection of proteins and domains by microscopy, using the same procedure I set up for membrane proteins in N. benthamiana. In addition, this specificity was compared with the structural properties of the proteins, such as charge and hydrophobicity. Unexpectedly, no correlation was found between these structural properties and GL specificity. Instead, it seems that this specificity is more influenced by the function of the proteins in GL biogenesis or by their arrival kinetics. Indeed, proteins that localize early on the surface of the monolayer show increased specificity. On the other hand, the affinity of proteins to GLs, i.e. the ability to remain associated with GLs, could not be quantified in this chassis, as the transient overproduction of GLs was not sufficient for this. A new chassis, a N. benthamiana stably overaccumulating GLs, therefore had to be produced. Compared with the WT, this chassis has 22 to 23 times more GLs. After isolation, these GLs were subjected to various washes, with increasingly stringent conditions, and the associated proteins were detected by biochemical techniques. This affinity assessment technique, initially based on the seed, was used for the first time in a leaf context. The results obtained for three proteins showed that the proteins were present as oligomers on the GLs and remained bound regardless of the stringency of the treatment. The results obtained previously were validated in C. sativa seeds, by tracing the production of a commercial growth factor protein, HsFGF2. These observations highlight that the arrival kinetics and functional role of proteins in GL biogenesis are determining factors for their specificity and affinity in GL addressing. This knowledge will help optimize the use of GLs and their associated proteins for biotechnological applications.
Director: Jean-Denis Faure - INRAE, IJPB, Versailles, "Dynamics and Engineering of Lipidic Compartments" DECLIC team
Co-Director: Chouaïb Meziadi, Core Biogenesis, Strasbourg
Co-supervisor: Marine Froissard - INRAE, IJPB, Versailles, "Dynamics and Engineering of Lipidic Compartments" DECLIC team
Jury members
> Juliette Salvaing (Rapportrice) - INRAE, Irig, Grenoble
> Alenka Čopič (Rapportrice) - CNRS, CRBM,Montpellier
> Christelle Lopez (Examinatrice) - INRAE, BIA, Nantes
> Michel Hernould (Examinateur) - Université Bordeaux, BFP, Villenave d'Ornon
Florent Fontaine is taking the doctoral student oath.
Research developed at the Institute Jean-Pierre Bourgin for Plant Sciences.
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