Engineering and Characterization of a Fluorescent Native-Like HIV-1 Envelope Glycoprotein Trimer

• Published in: Biomolecules (Basel, Switzerland) Vol. 5; no. 4; pp. 2919 - 2934
• Main Authors: Sliepen, Kwinten, van Montfort, Thijs, Ozorowski, Gabriel, Pritchard, Laura K, Crispin, Max, Ward, Andrew B, Sanders, Rogier W
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 23.10.2015; MDPI AG
• Subjects: Amino Acid Sequence; Gene Products, env - chemistry; Gene Products, env - genetics; Gene Products, env - metabolism; green fluorescent protein (GFP); Green Fluorescent Proteins - genetics; HEK293 Cells; HIV-1; HIV-1 Env; Humans; Molecular Sequence Data; protein engineering; Protein Multimerization; Protein Stability; Protein Transport; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SOSIP; superfolder GFP; SOSIP; superfolder GFP; protein engineering; green fluorescent protein (GFP); HIV-1 Env
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom5042919

Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein has facilitated major advances in the HIV-1 vaccine field, since it was the first Env immunogen that induced consistent neutralizing antibodies against a neutralization-resistant (tier 2) virus. Moreover, BG505 SOSIP.664 enabled elucidation of the atomic resolution structure of the Env trimer and facilitated the isolation and characterization of new broadly neutralizing antibodies against HIV-1. Here, we designed and characterized the BG505 SOSIP.664 trimer fused to fluorescent superfolder GFP (sfGFP), a GFP variant that allows efficient folding (BG505 SOSIP.664-sfGFP). Despite the presence of the sfGFP, the Env protein largely retained its morphology, antigenicity, glycan composition, and thermostability. In addition, we show that BG505 SOSIP.664-sfGFP can be used for fluorescence-based assays, such as flow cytometry.