De novo design of modular and tunable protein biosensors.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

De novo design of modular and tunable allosteric biosensors.

Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1 , but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2 . Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3 , we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Generation of a Human Monoclonal Antibody to Cross-Reactive Material 197 (CRM197) and Development of a Sandwich ELISA for CRM197 Conjugate Vaccines.

Cross-reactive material 197 (CRM197) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM197 and CRM197 conjugate vaccines, we generated a human anti-CRM197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (KD) of 3F9 for CRM197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM197. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was ＜1 ng/ml CRM197. In addition, the 3F9 antibody bound to the CRM197-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM197 and CRM197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM197 conjugate vaccines.

Generation and Characterization of a Neutralizing Human Monoclonal Antibody to Hepatitis B Virus PreS1 from a Phage-Displayed Human Synthetic Fab Library.

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

Construction and Characterization of an Anti-Hepatitis B Virus preS1 Humanized Antibody that Binds to the Essential Receptor Binding Site.

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.

Purification, crystallization and X-ray crystallographic studies on a putative methyltransferase, YtqB, from Bacillus subtilis.

S-Adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) catalyze the transfer of a methyl group from a SAM cofactor to specific substrate molecules, including small chemicals, proteins, DNAs and RNAs, and are required for various cellular functions, such as regulation of gene expression and biosynthesis of metabolites. Bacillus subtilis YtqB is a putative SAM-dependent MTase whose biological function has not been characterized. To provide biochemical and structural insights into the role of YtqB in bacteria, the recombinant YtqB protein was overexpressed in the Escherichia coli expression system and purified by chromatographic methods. YtqB crystals were obtained in PEG-containing conditions and diffracted to 1.68 Šresolution. The YtqB crystals belonged to space group P212121, with two molecules in the asymmetric unit.