High Yield, Site-Specific Coupling of N-Terminally Modified .beta.-Lactamase to a Proteolytically Derived Single-Sulfhydryl Murine Fab'

1994 ◽  
Vol 5 (6) ◽  
pp. 636-646 ◽  
Author(s):  
Stephen D. Mikolajczyk ◽  
Damon L. Meyer ◽  
James J. Starling ◽  
Kevin L. Law ◽  
Keith Rose ◽  
...  
Keyword(s):  
High Yield ◽  
Beta Lactamase ◽  
Site Specific

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

scholarly journals Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Pralow ◽  
Alexander Nikolay ◽  
Arnaud Leon ◽  
Yvonne Genzel ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Zika Virus ◽  
Animal Cell ◽  
Gradient Centrifugation ◽  
Viral Envelope ◽  
Protein Glycosylation ◽  
High Yield ◽  
Structural Protein ◽  
Site Specific ◽  
E Protein ◽  
The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2019 ◽  
Vol 141 (9) ◽  
pp. 3875-3884 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L. V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
High Yield ◽  
Site Specific ◽  
Specific Modification

scholarly journals Synthetic Elaboration of Native DNA by RASS (SENDR)

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Kyle W. Knouse ◽  
Julien C. Vantourout ◽  
Brittany Sanchez ◽  
Emily J. Sturgell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Solid Support ◽  
High Yield ◽  
Organic Media ◽  
Two Stage ◽  
Site Specific ◽  
Reversible Adsorption ◽  
Dna Conformations ◽  
Single Coupling ◽  
Stage Process

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

Synthetic Elaboration of Native DNA by RASS (SENDR)

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Kyle W. Knouse ◽  
Julien C. Vantourout ◽  
Brittany Sanchez ◽  
Emily J. Sturgell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Solid Support ◽  
High Yield ◽  
Organic Media ◽  
Two Stage ◽  
Site Specific ◽  
Reversible Adsorption ◽  
Dna Conformations ◽  
Single Coupling ◽  
Stage Process

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

Effects of precision potato planting using GPS-based cultivation

2017 ◽  
Vol 8 (2) ◽  
pp. 450-454
Author(s):  
Y. Reckleben ◽  
T. Grau ◽  
S. Schulz ◽  
H. G. Trumpf
Keyword(s):  
Soil Properties ◽  
Sandy Soil ◽  
Field Trials ◽  
High Yield ◽  
Site Specific ◽  
Heavy Soil ◽  
Specific Management

Site-specific management provides the ability to align the production intensity to demand and thus adjust the expenses to the necessary level. So it is possible to increase the proportion of marketable commodity in the normal sort–size of 40 mm to 60 mm. Planting distances adapted to the soil properties seem to achieve this objective. It is possible to further optimize the proportion of marketable commodity especially in the potato regions where irrigation and fertilization already contribute to a consistently high yield. Different planting distances on the soil sites by EM38 were tested in field trials. Planting distances of 31.50 cm in the row on the light (sandy) soil, 24.50 cm on middle and 27.50 cm on the heavy soil sites seems the best for these three years. There is a yield impact in total, as well as in the proportion of marketable commodity. Depending on the planting strategy, increases in income up to €153 per hectare can be obtained.

scholarly journals High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

2012 ◽  
Vol 418 (4) ◽  
pp. 652-656 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Karin V. Loscha ◽  
Kekini V. Kuppan ◽  
Choy Theng Loh ◽  
Nicholas E. Dixon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
High Yield ◽  
Site Specific ◽  
Free Protein ◽  
Cell Free Protein Synthesis
Sign in / Sign up

Export Citation Format

Share Document