Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy...

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Autores principales:	John W Lamppa, Margaret E Ackerman, Jennifer I Lai, Thomas C Scanlon, Karl E Griswold
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/70e3c1c343d9462c9be2bdfc4b106346
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spelling	oai:doaj.org-article:70e3c1c343d9462c9be2bdfc4b1063462021-11-18T06:58:51ZGenetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.1932-620310.1371/journal.pone.0017042https://doaj.org/article/70e3c1c343d9462c9be2bdfc4b1063462011-02-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21340021/?tool=EBIhttps://doaj.org/toc/1932-6203Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.John W LamppaMargaret E AckermanJennifer I LaiThomas C ScanlonKarl E GriswoldPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 2, p e17042 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q John W Lamppa Margaret E Ackerman Jennifer I Lai Thomas C Scanlon Karl E Griswold Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
description	Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.
format	article
author	John W Lamppa Margaret E Ackerman Jennifer I Lai Thomas C Scanlon Karl E Griswold
author_facet	John W Lamppa Margaret E Ackerman Jennifer I Lai Thomas C Scanlon Karl E Griswold
author_sort	John W Lamppa
title	Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
title_short	Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
title_full	Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
title_fullStr	Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
title_full_unstemmed	Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
title_sort	genetically engineered alginate lyase-peg conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/70e3c1c343d9462c9be2bdfc4b106346
work_keys_str_mv	AT johnwlamppa geneticallyengineeredalginatelyasepegconjugatesexhibitenhancedcatalyticfunctionandreducedimmunoreactivity AT margareteackerman geneticallyengineeredalginatelyasepegconjugatesexhibitenhancedcatalyticfunctionandreducedimmunoreactivity AT jenniferilai geneticallyengineeredalginatelyasepegconjugatesexhibitenhancedcatalyticfunctionandreducedimmunoreactivity AT thomascscanlon geneticallyengineeredalginatelyasepegconjugatesexhibitenhancedcatalyticfunctionandreducedimmunoreactivity AT karlegriswold geneticallyengineeredalginatelyasepegconjugatesexhibitenhancedcatalyticfunctionandreducedimmunoreactivity
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Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

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