Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

• Published in: Scientific reports Vol. 10; no. 1; p. 13323
• Main Authors: Tang, Yanyang, Saul, Justin, Nagaratnam, Nirupa, Martin-Garcia, Jose M, Fromme, Petra, Qiu, Ji, LaBaer, Joshua
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 07.08.2020; Nature Publishing Group UK
• Subjects: Animals; Baculoviridae - genetics; Baculoviridae - metabolism; Crystallography; Crystallography, X-Ray; Expression vectors; Gene Expression; Genetic Vectors; Insect cells; Kinases; Microscopy, Electron, Transmission; Protein expression; Protein purification; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Sf9 Cells; Spodoptera; X-ray crystallography
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-70163-2

Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.