A fluorometric assay for pepsin and pepsinogen was developed using enhanced green fluorescent protein (EGFP) as a substrate. Acid denaturation of EGFP resulted in a complete loss of fluorescence that was completely reversible on neutralization. In the proteolytic assay procedure, acid-denatured EGFP was digested by pepsin or activated pepsinogen. After neutralization, the remaining amount of un...

BACKGROUND Mild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies. RESULTS Here we presented an efficient method for mild solubilization of inclusion bodies by using a...

The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various condition...

The vasa gene (vas) is essential for germline development in Drosophila melanogaster. Zygotic vas is expressed in pole cells earlier than any other pole cell-expressing genes thus far identified, and VAS protein is continuously present in germline cells throughout development. Here, we report the identification of a regulatory region that directs germline-specific vas expression. A genomic frag...

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly eff...

Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug sel...

Trypanosoma congolense epimastigote forms (EMFs) adhere to the tsetse fly proboscis, proliferate, and differentiate into animal-infective metacyclic forms (MCFs). This differentiation step, called metacyclogenesis, is indispensable for the cyclical transmission of the parasite. Although an in vitro metacyclogenesis culture system was established several decades ago, few genetic tools have been ...

There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73:51-54, 1999). We established a highly sensitive assay to determine frequencie...

A new rapid method for titrating baculovirus stocks has been developed using a novel cell line Sf-9 Easy Titer (Sf-9ET). The Sf-9ET cell line has been transfected with plasmid DNA containing the enhanced green fluorescent protein (eGFP) gene under the control of the baculovirus polyhedrin promoter. When used in the titration assay, the Sf-9ET cells turn green when they are infected with baculov...

A Na(+)/H(+) antiporter gene (CvNHA1) was cloned from the salt-tolerant yeast Candida versatilis. CvNHA1 encodes an antiporter with a typical yeast plasma membrane Na(+)/H(+) antiporter structure. Transcription of CvNHA1 in C. versatilis cells was dependent on the salinity of the culture. When CvNHA1 was expressed in salt-sensitive Saccharomyces cerevisiae cells, increased salt-tolerance was ob...