A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-ß protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.

Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins: From Expression Screening for Soluble Constructs to Protein Quality Control.

Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.

DNA recognition by retinoic acid nuclear receptors.

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) to regulate gene expression. The heterodimer recognizes the genome via a large and diverse repertoire of DNA response elements. Assessing the binding mode of RAR and RXR with various DNA response elements is important for understanding how they select their binding site and how DNA sequence and topology allosterically regulate RAR function. A number of complementary assays are often employed for analysis of the binding mode. To biochemically and structurally characterize RAR and RXR-DNA complexes, we describe how to express and purify RAR and RXR-DNA binding domains (DBDs) and multidomain constructs. We also describe the use of electrospray ionization mass spectrometry (ESI MS) and isothermal titration calorimetry (ITC) that give information about stoichiometry and binding affinity, as well as our approaches for co-crystallization of RAR and RXR DBDs with DNA.