ABSTRACT
The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 µm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
Subject(s)
Microscopy, Electron, Transmission/methods , Nanoparticles/ultrastructure , Proteins/ultrastructure , Recombinant Proteins/ultrastructure , Animals , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Nanoparticles/chemistry , Proteins/chemistry , Proteins/genetics , Recombinant Proteins/chemistry , Reproducibility of Results , Sf9 CellsABSTRACT
To date, the phage display methodology is a great choice in clinical diagnostic field. Using phage display is possible design and choose peptides and proteins that are expressed on filamentous phage surface. So, these extremely reactive and specific biomolecules can be used in several diagnostic tests. The advantages of phage display over other methodologies have been shown because of its successful results in biomedical and clinical research. The more important reaches of phage display in clinical diagnostic and the way that has been applied to different diseases will be described in this review.
Subject(s)
Diagnosis , Peptide Library , Antibodies , Gene Library , Genomic Library , HumansABSTRACT
Antibody-dependent cellular cytotoxicity (ADCC) is a host defense mechanism in which Fc receptor-bearing effector cells in combination with antigen-specific antibodies recognize and kill antigen-expressing target cells. The authors previously described a murine monoclonal antibody (MAb-ID6) that mediated ADCC activity against HIV-infected cells. It was demonstrated that the specificity of MAb-ID6 maps to the first 204 amino acids of gp120; however, the exact epitope was not identified. In the present work, by screening phage display libraries with MAb-ID6, the authors have mapped the corresponding epitope to amino acids 86-100 (HIV-1 gp120 sequence). This epitope lies within the C1 region of gp120 and is highly conserved among all subtypes and circulating recombinant forms of HIV-1. Thus, these phage mimotopes of C1 may serve as components of a vaccine for the induction of gp120-specific antibodies mimicking MAb-ID6.
