2019-nCoV is the causative agent of the serious, still ongoing, worldwide coronavirus disease (COVID-19) pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease (HRV3C) cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA immobilized metal ion affinity chromatography (IMAC) purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

Introduction of specific point mutations has been an effective strategy in enhancing the thermal stability in detergents that aid the purification of mono-dispersed G-protein coupled receptors (GPCRs). Our previous work showed that a specific residue position on transmembrane helix 6 (TM6) in class A GPCRs consistently yields thermostable mutants. The crystal structure of human chemokine receptor CCR5 also showed increased thermostability at two positions, A233D6.33 and K303E7.59. With the goal of testing the transferability of these two thermostabilizing mutations in the other chemokine receptors, we tested the mutations A237D6.33 and R307E7.59 in human CCR3 for thermostability and aggregation properties in DDM detergent solution. Interestingly, the double mutant exhibited a 6–10 fold decrease in the aggregation propensity of the wild type protein. This is in stark contrast to the two single mutants whose aggregation properties resemble more to the wild type (WT). Moreover, Unlike in CCR5, the two single mutants separately showed no increase in thermostability compared to the wild type CCR3, while the double mutant A237D6.33/R307E7.59 confers an increase of 2.6°C in the melting temperature compared to the WT. Extensive all-atom molecular dynamics (MD) simulations in detergent micelles show that a salt bridge network between transmembrane helices TM3, TM6 and TM7 that is absent in the two single mutants confers stability in the double mutant. Free energy surface of the double mutant shows conformational homogeneity compared to the single mutants. An annular n-dodecyl maltoside (DDM) detergent layer packs tighter to the hydrophobic surface of the double mutant CCR3 compared to the single mutants providing additional stability. The purification of other C-C chemokine receptors lacking such stabilizing residues may benefit from the incorporation of these two point mutations in appropriate TM regions.

Highlights d Three crystal structures and a cryo-EM structure of engineered fusion proteins d Rigid ER/K helix is used as a modular fusion protein linker d Crystal structure of a high-affinity calmodulin-binding peptide/calmodulin complex d Rigid fusion approach for structural biology and protein nanotechnology