Subcellular compartmentalization enables eukaryotic cells to carry out complex, multistep, or energetically unfavorable reactions with high efficiency by preventing side reactions and creating chemical gradients. In synthetic biology, functionalized particles enclosed by a lipid bilayer mimic this compartmentalization, offering similar advantages. These particles enhance the efficient utilization of substrates, including primary metabolites or even toxic compounds. In this study, enveloped virus-like particles (VLPs) were produced in the yeast Saccharomyces cerevisiae by heterologous expression of the HIV-1 Gag polyprotein, controlled under the inducible gal1 promoter. Purification and enrichment of VLPs were performed by density gradient ultracentrifugation. We show that the VLPs can be efficiently loaded with proteins of interest, such as lactate dehydrogenase (LDH) and green fluorescent protein (GFP), by post-translational fusion to the Gag protein. We also confirmed that the lipid bilayer serves as a protective barrier for the proteins encapsulated within the VLPs, as demonstrated through protease digestion experiments. The efficiency of the loading system was analyzed using nanoflow cytometry and proteomics analysis. Additionally, proteomics data revealed an enrichment of membrane proteins in the purified VLP samples. Future applications could leverage this system for substrate and product transport, as well as for the creation of (electro-)chemical gradients to drive diverse transport reactions. With further optimization, functionalized VLPs hold the promise of serving as a cell-free production platform, combining the benefits of compartmentalization with the added protection of encapsulated enzymes.