Generation of Tetracycline-Inducible Mammalian Cell Lines by Flow Cytometry for Improved Overproduction of Membrane Proteins

2016 ◽  
pp. 63-78 ◽  
Author(s):  
Juni Andréll ◽  
Patricia C. Edwards ◽  
Fan Zhang ◽  
Maria Daly ◽  
Christopher G. Tate
Keyword(s):  
Flow Cytometry ◽  
Membrane Proteins ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cell Lines

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.

RAS p21 and other Gn proteins are detected in mammalian cell lines by [γ-35S]GTPγS binding

1989 ◽  
Vol 159 (3) ◽  
pp. 1269-1274 ◽  
Author(s):  
J.G. Comerford ◽  
J.R. Gibson ◽  
A.P. Dawson ◽  
I. Gibson
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cell Lines ◽  
Gtpγs Binding ◽  
Ras P21

scholarly journals Protocol to identify centrosome-associated transcription factors during mitosis in mammalian cell lines

2021 ◽  
Vol 2 (3) ◽  
pp. 100495
Author(s):  
Guobin Xie ◽  
Yuqi Zhou ◽  
Mingxuan Du ◽  
Qiqiang Wang ◽  
Jiajin Yi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cell Lines
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