Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins
| العنوان: | Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins |
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| المؤلفون: | Oliver M. Eder, Stewart D. Nuttall, Albert Ardevol, Thomas S. Peat, Janet Newman, Alessandro T. Caputo, Heleen Pothuis, Timothy E. Adams, Hana Bereznakova |
| المصدر: | Scientific Reports, Vol 11, Iss 1, Pp 1-11 (2021) Scientific Reports |
| بيانات النشر: | Nature Portfolio, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | 0301 basic medicine, Expression systems, Science, Mutant, Protein design, Biologics, Crystallography, X-Ray, Biochemistry, Article, Cell Line, law.invention, 03 medical and health sciences, chemistry.chemical_compound, Acetyl Coenzyme A, Acetyltransferases, law, Catalytic Domain, Animals, Humans, X-ray crystallography, chemistry.chemical_classification, Multidisciplinary, 030102 biochemistry & molecular biology, Chemistry, Acetylation, Transfection, Recombinant Proteins, Streptomyces, Enzymes, HEK293 Cells, 030104 developmental biology, Enzyme, Gene Expression Regulation, Puromycin, Mutation, Recombinant DNA, Medicine, Target protein, Structural biology, Function (biology) |
| الوصف: | 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. |
| اللغة: | English |
| تدمد: | 2045-2322 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::541e76503b7a59d18f3c3f108957c3cd https://doaj.org/article/e3fd4beb677e4374affab46d03fe0260 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....541e76503b7a59d18f3c3f108957c3cd |
| قاعدة البيانات: | OpenAIRE |
Find this article in full text from Springer Verlag. | تدمد: | 20452322 |
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