Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Abstract Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related...

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Autores principales:	Alessandro T. Caputo, Oliver M. Eder, Hana Bereznakova, Heleen Pothuis, Albert Ardevol, Janet Newman, Stewart Nuttall, Thomas S. Peat, Timothy E. Adams
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e3fd4beb677e4374affab46d03fe0260
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spelling	oai:doaj.org-article:e3fd4beb677e4374affab46d03fe02602021-12-02T13:34:57ZStructure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins10.1038/s41598-021-84551-92045-2322https://doaj.org/article/e3fd4beb677e4374affab46d03fe02602021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84551-9https://doaj.org/toc/2045-2322Abstract Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.Alessandro T. CaputoOliver M. EderHana BereznakovaHeleen PothuisAlbert ArdevolJanet NewmanStewart NuttallThomas S. PeatTimothy E. AdamsNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alessandro T. Caputo Oliver M. Eder Hana Bereznakova Heleen Pothuis Albert Ardevol Janet Newman Stewart Nuttall Thomas S. Peat Timothy E. Adams Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
description	Abstract Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.
format	article
author	Alessandro T. Caputo Oliver M. Eder Hana Bereznakova Heleen Pothuis Albert Ardevol Janet Newman Stewart Nuttall Thomas S. Peat Timothy E. Adams
author_facet	Alessandro T. Caputo Oliver M. Eder Hana Bereznakova Heleen Pothuis Albert Ardevol Janet Newman Stewart Nuttall Thomas S. Peat Timothy E. Adams
author_sort	Alessandro T. Caputo
title	Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
title_short	Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
title_full	Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
title_fullStr	Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
title_full_unstemmed	Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
title_sort	structure-guided selection of puromycin n-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e3fd4beb677e4374affab46d03fe0260
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Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

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