| http://purl.uniprot.org/citations/33664348 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
| http://purl.uniprot.org/citations/33664348 | http://www.w3.org/2000/01/rdf-schema#comment | "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."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.org/dc/terms/identifier | "doi:10.1038/s41598-021-84551-9"xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Newman J."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Peat T.S."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Ardevol A."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Caputo A.T."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Nuttall S."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Adams T.E."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Bereznakova H."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Eder O.M."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/author | "Pothuis H."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/date | "2021"xsd:gYear |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/name | "Sci Rep"xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/pages | "5247"xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/title | "Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins."xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://purl.uniprot.org/core/volume | "11"xsd:string |
| http://purl.uniprot.org/citations/33664348 | http://www.w3.org/2004/02/skos/core#exactMatch | http://purl.uniprot.org/pubmed/33664348 |
| http://purl.uniprot.org/citations/33664348 | http://xmlns.com/foaf/0.1/primaryTopicOf | https://pubmed.ncbi.nlm.nih.gov/33664348 |
| http://purl.uniprot.org/uniprot/#_P13249-mappedCitation-33664348 | http://www.w3.org/1999/02/22-rdf-syntax-ns#object | http://purl.uniprot.org/citations/33664348 |
| http://purl.uniprot.org/uniprot/P13249 | http://purl.uniprot.org/core/mappedCitation | http://purl.uniprot.org/citations/33664348 |