Roles and Functions of MFG-E8 as Carrier Proteins in Exosomes

doi:10.13560/j.cnki.biotech.bull.1985.2021-0814

Abstract

Abstract:

The objectives of this work are to construct the recombinant plasmids of full-length and truncated MFG-E8（milk fat globule epidermal growth factor 8）,to analyze whether each truncated protein can bring the target protein to exosomes determine their positions,for exploring the function of each domain of MFG-E8 and the efficiency as carrier proteins in exosomes. Polymerase chain reaction（PCR）was used to amplify the MFG-E8 and its truncated genes with signal peptide and fused with EGFP gene to construct 7 recombinant plasmids. Then,the plasmids were transfected into HEK293F cells using PEI. Western blot and laser scanning confocal microscope（LSCM）were to detect the proteins expressions and intracellular locations of MFG-E8 and its truncated recombinant proteins,respectively. Meanwhile,the exosomes secreted by the transfected cells were extracted and the recombinant proteins in the exosomes were detected by Western blot and electron microscopic. Finally,the exosomes harbored recombinant proteins were transfected into HEK293T cells and the results of exosomes bringing the recombinant proteins into receptor cells were observed by fluorescence microscopy. Results showed that MFG-E8 and its truncated plasmids were constructed successfully and expressed in HEK293F cells. All recombinant proteins were expressed in the cells,of them the expression of EGF-L was the highest,while the lowest for C2. Only EGF-L,C1 and EC1 were detected in the cell supernatant. MFG-E8,EC1 and EC2 were detected in the exosomes secreted by the transfected cells. With these exosomes containing 3 proteins transfected into HEK293T cells,the fluorescence was observed in all three groups of cells,of which the exosome from MFG-E8-EGFP was the most effective. In conclusion,MFG-E8 can be used as a carrier to bring the proteins of interest to the exosome membrane,and its C1 and C2 domains are crucial to this process. The presence of EGF-L may promote protein expression.

Key words: milk fat globule-epidermal growth factor VIII（MFG-E8）, truncated protein, exosome, carrier protein, function of domain

LI Zhi-kang, LIU Chen-xue-xuan, TAN Chu-min, XIONG Sheng, XIE Qiu-ling. Roles and Functions of MFG-E8 as Carrier Proteins in Exosomes[J]. Biotechnology Bulletin, 2022, 38(4): 288-294.

Figures/Tables 6

Fig. 1 Structure of MFG-E8-EGFP and its truncated forms as well as gel electrophoresis plots for identification by enzyme digestion A：Schematic diagram of the recombinant plasmids of MFG-E8-EGFP and its truncated forms. B：Gel electrophoresis plots for identifying the recombinant plasmids by enzyme digestion

Fig. 2 Detection of the transient efficiency and expression of the recombinant plasmids A：Detection of the transient efficiency of the recombinant plasmids in HEK293 cells by flow cytometry. B：Detection of target protein expression in HEK293 cells by Western blot. C：Detection of target protein expression in culture supernatant by Western blot

Fig. 3 Location detection of MFG-E8 and its truncated pr-oteins in HEK293 cells by laser confocal microscopy

Fig. 4 Transmission electron micrograph and size distribu-tions of exosomes A：Transmission electron micrograph of exosomes,scale bar,100 nm. B：Size distributions of exosomes on NTA measurement

Fig. 5 Identification of EGFP in exosomes A：Western blot detection of MFG-E8-EGFP and its truncated ones in exosomes.B：Transmission electron microscopy images of exosomes derived from HEK293F cell transfected with pCMV-MFG-E8-EGFP（b）,pCMV-EC1-EGFP（c）or pCMV-C1C2-EGFP（d）. Scale bars,100 nm

Fig. 6 Fluorescence images showing the effect of exosomes transfection

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