Sulfhydryl-Specific PEGylation of Phosphotriesterase Cysteine Mutants for Organophosphate Detoxification

Gurdip K Daffu; Patricia Lopez; Francine Katz; Michael Vinogradov; Chang-Guo Zhan; Donald W Landry; Joanne Macdonald

doi:10.1093/protein/gzv036

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Sulfhydryl-Specific PEGylation of Phosphotriesterase Cysteine Mutants for Organophosphate Detoxification

Journal article

Peer reviewed

Sulfhydryl-Specific PEGylation of Phosphotriesterase Cysteine Mutants for Organophosphate Detoxification

Gurdip K Daffu, Patricia Lopez, Francine Katz, Michael Vinogradov, Chang-Guo Zhan, Donald W Landry and Joanne Macdonald

Protein Engineering Design and Selection, Vol.28(11), pp.501-506

2015

DOI: https://doi.org/10.1093/protein/gzv036

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Abstract

fluorescein 5-maleimide

paraoxon

phosphotriesterase

polyethylene glycol

site-directed mutagenesis

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

Details

Title: Sulfhydryl-Specific PEGylation of Phosphotriesterase Cysteine Mutants for Organophosphate Detoxification
Authors: Gurdip K Daffu (Author) - Columbia University Medical Centre, United States
Patricia Lopez (Author) - Columbia University Medical Centre, United States
Francine Katz (Author) - Columbia University Medical Centre, United States
Michael Vinogradov (Author) - Columbia University Medical Centre, United States
Chang-Guo Zhan (Author) - University of Kentucky, United States
Donald W Landry (Author) - Columbia University Medical Centre, United States
Joanne Macdonald (Author) - University of the Sunshine Coast - Faculty of Science, Health, Education and Engineering
Publication details: Protein Engineering Design and Selection, Vol.28(11), pp.501-506
Publisher: Oxford University Press
Date published: 2015
DOI: 10.1093/protein/gzv036
ISSN: 1741-0126
Organisation Unit: School of Science and Engineering - Legacy; University of the Sunshine Coast, Queensland; School of Science, Technology and Engineering; Centre for Bioinnovation
Language: English
Record Identifier: 99449285802621
Output Type: Journal article

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Collaboration types: Domestic collaboration; International collaboration
Web Of Science research areas: Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology

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