scholarly journals Expression of frog virus 3 genes is impaired in mammalian cell lines

2008 ◽  
Vol 5 (1) ◽  
pp. 83 ◽  
Author(s):  
Heather E Eaton ◽  
Julie Metcalf ◽  
Craig R Brunetti
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  

AbstractPuromycin 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.


1989 ◽  
Vol 159 (3) ◽  
pp. 1269-1274 ◽  
Author(s):  
J.G. Comerford ◽  
J.R. Gibson ◽  
A.P. Dawson ◽  
I. Gibson

2021 ◽  
Vol 2 (3) ◽  
pp. 100495
Author(s):  
Guobin Xie ◽  
Yuqi Zhou ◽  
Mingxuan Du ◽  
Qiqiang Wang ◽  
Jiajin Yi ◽  
...  

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