The incredible potential for fluorescent proteins to revolutionize biology has inspired the development of a variety of design strategies to address an equally broad range of photophysical characteristics, depending on potential applications. Of these, fluorescent proteins that simultaneously exhibit high quantum yield, red-shifted emission, and wide separation between excitation and emission wavelengths (Large Stokes Shift, LSS) are rare. The pursuit of LSS systems has led to the formation of a complex, obtained from the marriage of a rationally engineered protein (human cellular retinol binding protein II, hCRBPII) and different fluorogenic molecules, capable of supporting photobase activity. The large increase in basicity upon photoexcitation leads to protonation of the fluorophore in the excited state, dramatically red-shifting its emission, leading to an LSS protein/fluorophore complex. Essential for selective photobase activity is the intimate involvement of the target protein structure and sequence that enables Excited State Proton Transfer (ESPT). The potential power and usefulness of the strategy was demonstrated in live cell imaging of human cell lines.
Vaccines are critical tools to treat and prevent diseases.
For
an effective conjugate vaccine, the carrier is crucial, but few carriers
are available for clinical applications. In addition, a drawback of
current protein carriers is that high levels of antibodies against
the carrier are induced by the conjugate vaccine, which are known
to interfere with the immune responses against the target antigen.
To overcome these challenges, we obtained the near atomic resolution
crystal structure of an emerging protein carrier, i.e., the bacteriophage
Qβ virus like particle. On the basis of the detailed structural
information, novel mutants of bacteriophage Qβ (mQβ) have
been designed, which upon conjugation with tumor associated carbohydrate
antigens (TACAs), a class of important tumor antigens, elicited powerful
anti-TACA IgG responses and yet produced lower levels of anticarrier
antibodies as compared to those from the wild type Qβ-TACA conjugates.
In a therapeutic model against an aggressive breast cancer in mice,
100% unimmunized mice succumbed to tumors in just 12 days even with
chemotherapy. In contrast, 80% of mice immunized with the mQβ-TACA
conjugate were completely free from tumors. Besides TACAs, to aid
in the development of vaccines to protect against COVID-19, the mQβ
based conjugate vaccine has been shown to induce high levels of IgG
antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating
its generality. Thus, mQβ is a promising next-generation carrier
platform for conjugate vaccines, and structure-based rational design
is a powerful strategy to develop new vaccine carriers.
With the infection rate of Bordetella pertussis at a 60‐year high, there is an urgent need for new anti‐pertussis vaccines. The lipopolysaccharide (LPS) of B. pertussis is an attractive antigen for vaccine development. With the presence of multiple rare sugars and unusual glycosyl linkages, the B. pertussis LPS is a highly challenging synthetic target. In this work, aided by molecular dynamics simulation and modeling, a pertussis‐LPS‐like pentasaccharide was chemically synthesized for the first time. The pentasaccharide was conjugated with a powerful carrier, bacteriophage Qβ, as a vaccine candidate. Immunization of mice with the conjugate induced robust anti‐glycan IgG responses with IgG titers reaching several million enzyme‐linked immunosorbent assay (ELISA) units. The antibodies generated were long lasting and boostable and could recognize multiple clinical strains of B. pertussis, highlighting the potential of Qβ‐glycan as a new anti‐pertussis vaccine.
FR-1V, a fluorene-based aldehydic chromophore, binds its target protein as an imine to yield a highly bathochromic pigment, CF-2, a prototypic protein-dye tagging system whose NIR emission can be spatiotemporally switched ON by rapid UV-light activation. This is achieved through photoisomerization of the imine and its subsequent protonation. We demonstrate a nowash protocol for live cell imaging of subcellular compartments in a variety of mammalian cell lines with minimal fluorescence background.
The conjugate of a synthetic Salmonella Enteritidis tetrasaccharide with bacteriophage Qβ induced powerful anti-glycan IgG responses for complete protection from lethal challenges of bacteria.
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