scholarly journals A Novel High-Throughput Protein Engineering Platform

2017 ◽  
Author(s):  
Thomas M Baer
Keyword(s):  
Protein Engineering ◽  
High Throughput

scholarly journals Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

ACS Omega ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. 1498-1508 ◽  
Author(s):  
Xue W. Diefenbach ◽  
Iman Farasat ◽  
Erik D. Guetschow ◽  
Christopher J. Welch ◽  
Robert T. Kennedy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Engineering ◽  
High Throughput ◽  
Droplet Microfluidics

scholarly journals High-Throughput Thermal Scanning: A General, Rapid Dye-Binding Thermal Shift Screen for Protein Engineering

2009 ◽  
Vol 131 (11) ◽  
pp. 3794-3795 ◽  
Author(s):  
Jason J. Lavinder ◽  
Sanjay B. Hari ◽  
Brandon J. Sullivan ◽  
Thomas J. Magliery
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
Dye Binding ◽  
Thermal Shift

Confocal and Conventional Fluorescence-Based High Throughput Screening in Protein Engineering

2011 ◽  
pp. 713-751
Author(s):  
Ulrich Haupts ◽  
Oliver Hesse ◽  
Michael Strerath ◽  
Peter J. Walla ◽  
Wayne M. Coco
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Droplet Microfluidics-Enabled High-Throughput Screening for Protein Engineering

Micromachines ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 734 ◽  
Author(s):  
Lindong Weng ◽  
James E. Spoonamore
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
High Throughput ◽  
Protein Design ◽  
High Throughput Screening ◽  
Fluid Phase ◽  
Droplet Microfluidics ◽  
New Paradigm ◽  
Wide Range ◽  
Challenges And Opportunities

Protein engineering—the process of developing useful or valuable proteins—has successfully created a wide range of proteins tailored to specific agricultural, industrial, and biomedical applications. Protein engineering may rely on rational techniques informed by structural models, phylogenic information, or computational methods or it may rely upon random techniques such as chemical mutation, DNA shuffling, error prone polymerase chain reaction (PCR), etc. The increasing capabilities of rational protein design coupled to the rapid production of large variant libraries have seriously challenged the capacity of traditional screening and selection techniques. Similarly, random approaches based on directed evolution, which relies on the Darwinian principles of mutation and selection to steer proteins toward desired traits, also requires the screening of very large libraries of mutants to be truly effective. For either rational or random approaches, the highest possible screening throughput facilitates efficient protein engineering strategies. In the last decade, high-throughput screening (HTS) for protein engineering has been leveraging the emerging technologies of droplet microfluidics. Droplet microfluidics, featuring controlled formation and manipulation of nano- to femtoliter droplets of one fluid phase in another, has presented a new paradigm for screening, providing increased throughput, reduced reagent volume, and scalability. We review here the recent droplet microfluidics-based HTS systems developed for protein engineering, particularly directed evolution. The current review can also serve as a tutorial guide for protein engineers and molecular biologists who need a droplet microfluidics-based HTS system for their specific applications but may not have prior knowledge about microfluidics. In the end, several challenges and opportunities are identified to motivate the continued innovation of microfluidics with implications for protein engineering.

scholarly journals High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Jonathan T. Sullivan ◽  
Chidananda Sulli ◽  
Alberto Nilo ◽  
Anila Yasmeen ◽  
Gabriel Ozorowski ◽  
...  
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
Envelope Glycoprotein ◽  
High Throughput Screening ◽  
Neutralizing Antibody ◽  
Structural Basis ◽  
Vaccine Candidates ◽  
Clade B ◽  
Broadly Neutralizing Antibody ◽  
Hiv 1

ABSTRACT Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies. IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

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