ArticleScientific reports2021
Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 9 citations in OpenAlex.
- Bacterial Glutamine Synthetases as a Novel Metabolic Selection Marker to Improve CHO Cell Culture Performance Through Selection Stringency Modulation.Biotechnology journal · 2025Article
- Recombinant Cell-Permeable PuromycinJournal of microbiology and biotechnology · 2025Article
- Anti-PD-L1 blockade facilitates antitumor effects of radiofrequency ablation by improving tumor immune microenvironment in hepatocellular carcinoma.Apoptosis : an international journal on programmed cell death · 2025Article
- Mining Translation Inhibitors by a Unique Peptidyl-Aminonucleoside Synthetase Reveals Cystocin Biosynthesis and Self-Resistance.International journal of molecular sciences · 2024Article
- The soluble (pro)renin receptor promotes a preeclampsia-like phenotype both in vitro and in vivo.Hypertension research : official journal of the Japanese Society of Hypertension · 2024Article
- miR-186 induces tetraploidy in arsenic exposed human keratinocytes.Ecotoxicology and environmental safety · 2023Article
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Authors and funding
9 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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