efficient-site-specific-antibody-labeling-strain-promoted-azide

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Sogang University

sogang university

This paper proposes a new measurement system for measuring the refractive power of spherical and sphero-cylindrical lenses with a six-point light source, which is composed of a light emitting diode and a six-hole pattern aperture, and magnification ellipse fitting method. The position of the six light sources is changed into a circular or elliptical form subjected to the lens refractive power and meridian rotation angle. The magnification ellipse fitting method calculates the lens refractive power based on the ellipse equation with magnifications that are the ratios between initial diagonal lengths and measured diagonal lengths of the conjugated light sources changed by the target lens. The refractive powers of the spherical and sphero-cylindrical lenses certified in the Korea Research Institute of Standard and Science were measured to verify the measurement performance. The proposed method is estimated to have a repeatability of +/-0.01 D and an error value below 1%.

Measurement system of the refractive power of spherical and sphero-cylindrical lenses with the magnification ellipse fitting method.

We fabricated a novel in-line conductive atomic force microscopy (C-AFM), which can analyze the resistive failures and examine process variance with an exact-positioning capability across the whole wafer scale in in-line DRAM fabrication process. Using this in-line C-AFM, we introduced a new, non-destructive diagnosis for resistive failure in mobile DRAM structures. Specially, we focused on the self-aligned contact (SAC) process, because the failure of the SAC process is one of the dominant factors that induces the degradation of yield performance, and is a physically invisible defect. We successfully suggested the accurate pass mark for resistive-failure screening in the fabrication of SAC structures and established that the cause of SAC failures is the bottom silicon oxide layer. Through the accurate pass mark for the SAC process configured by the in-line C-AFM analyses, we secured a good potential method for preventing the yield loss caused by failures in DRAM fabrication.

Fast, exact, and non-destructive diagnoses of contact failures in nano-scale semiconductor device using conductive AFM.

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.

A fluorescence-based glycosyltransferase assay for high-throughput screening.

Protein-nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein-DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein-DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid.

The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

Genetic incorporation of recycled unnatural amino acids.

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

Wooseok Ko

Department of Chemistry

Sogang University

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Wooseok Ko

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry

Sogang University

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Department of Chemistry