Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation.

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization.

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors.

Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting.

Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. In these experiments, if cell viability is compromised by the sort conditions and/or expression of the target protein(s), rescue PCR provides an alternative to culturing but requires re-cloning and can introduce amplification bias. We have optimized a simple protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for subsequent transformation.

Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins.

Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal β-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial.

Influence of umbelliferone on membrane-bound ATPases in streptozotocin-induced diabetic rats.

The activities of membrane-bound ATPases are altered both in erythrocytes and tissues of streptozotocin (STZ)-induced diabetic rats and diabetic patients. Umbelliferone (UMB), a natural antioxidant, is a benzopyrone occurring in nature, and it is present in the fruits of golden apple (Aegle marmelos Correa) and bitter orange (Citrus aurantium). Earlier we evaluated and reported the effect of UMB on plasma insulin and glucose, and this study was designed to evaluate the effect of umbelliferone on membrane-bound ATPases in erythrocytes and tissues (liver, kidney and heart) of STZ-induced diabetic rats.

Effect of umbelliferone on tail tendon collagen and haemostatic function in streptozotocin-diabetic rats.

Diabetes mellitus is known to affect collagen in various tissues. Umbelliferone (7-hydroxycoumarin), a natural antioxidant and benzopyrone, is found in golden apple (Aegle marmelos Correa) and bitter orange (Citrus aurantium). Plant-derived phenolic coumarins have been shown to act as dietary antioxidants.

Protective effect of Umbelliferone on membranous fatty acid composition in streptozotocin-induced diabetic rats.

Umbelliferone (UMB), a natural antioxidant, is benzopyrone in nature, and it is present in the fruits of golden apple and bitter orange. Earlier we evaluated and reported the effect of Umbelliferone on antidiabetic, antioxidant and antihyperlipidemic properties, and this study was designed to evaluate the effect of Umbelliferone on membrane fatty acid composition and histopathology of liver and kidney of control and streptozotocin (STZ) diabetic rats. Male albino Wistar rats (180-200 g) were made diabetic by an intraperitonial administration of STZ (40 mg/kg).