scholarly journals Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

2002 ◽  
Vol 99 (21) ◽  
pp. 13413-13418 ◽  
Author(s):  
P. J. Reeves ◽  
J.-M. Kim ◽  
H. G. Khorana
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Structure And Function ◽  
High Level Expression ◽  
Mammalian Cell Lines ◽  
High Level ◽  
And Function ◽  
Tetracycline Inducible System

Humanc-fos promoter mediates high-level, inducible expression in various mammalian cell lines

2003 ◽  
Vol 81 (7) ◽  
pp. 848-854 ◽  
Author(s):  
Jing-Xiu Bi ◽  
Petra Buhr ◽  
An-Ping Zeng ◽  
Manfred Wirth
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Inducible Expression ◽  
Mammalian Cell Lines ◽  
High Level

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

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.

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