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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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-03-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-84551-9 |
| _version_ | 1818400711118946304 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T07:40:55Z |
| format | Article |
| id | doaj.art-e3fd4beb677e4374affab46d03fe0260 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T07:40:55Z |
| publishDate | 2021-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e3fd4beb677e4374affab46d03fe02602022-12-21T23:11:02ZengNature PortfolioScientific Reports2045-23222021-03-0111111110.1038/s41598-021-84551-9Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteinsAlessandro T. Caputo0Oliver M. Eder1Hana Bereznakova2Heleen Pothuis3Albert Ardevol4Janet Newman5Stewart Nuttall6Thomas S. Peat7Timothy E. Adams8Biomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationBiomedical Manufacturing, Commonwealth Scientific and Industrial Research OrganisationAbstract 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.https://doi.org/10.1038/s41598-021-84551-9 |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-021-84551-9 |
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