ABSTRACT
Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models.
Subject(s)
DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Lactic Acid/metabolism , Nanoparticles/metabolism , Polymers/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Drosophila melanogaster/physiology , Inclusion Bodies , Lactic Acid/chemistry , Nanoparticles/chemistry , Polyesters , Polymers/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Surface Properties , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/isolation & purificationABSTRACT
Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.
